JP7503539B2 - Assessment of host RNA using isothermal amplification and relative abundance - Google Patents

Description

CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 62/733,517, filed September 19, 2018, the entirety of which is incorporated herein by reference.

The present disclosure relates to a method for estimating a diagnostic score. More specifically, the present disclosure relates to a method for estimating a diagnostic score using real-time quantitative isothermal amplification. A test sample includes a first target nucleic acid and a second target nucleic acid. A first relative abundance value of the first target nucleic acid and a second relative abundance value of the second target nucleic acid are estimated by performing a real-time quantitative isothermal amplification assay. A diagnostic score is estimated based on the first relative abundance value and the second relative abundance value.

Quantification of target genes that are present at low abundance (e.g., low copy number) among other genes and nucleic acids in a sample can be difficult. Current detection methods often involve amplification (e.g., polymerase chain reaction (PCR)) of the gene or the product associated with the transcription of the gene (e.g., mRNA). With the advent of reverse transcriptase (RT)-PCR, RNA molecules can be specifically targeted to form cDNA molecules. The resulting cDNA molecules themselves can be used as templates for amplification, facilitating improved detection of the target gene or gene product in a sample. Isothermal amplification methods allow for faster amplification rates and lower instrument complexity. However, isothermal amplification techniques such as real-time quantitative loop-mediated isothermal amplification (real-time qLAMP)-based methods may not reliably quantify target nucleic acids with fewer than 1,000 copies of the target nucleic acid for mammalian RNA targets (see Nixon et al., (2014), Bimolecular Detection and Quantitation, 2:4-10).

Improved diagnosis of acute infections (such as bacterial and viral) could reduce morbidity and mortality. However, methods to detect acute infections are deficient in terms of specificity, sensitivity and speed. Although some amplification techniques can detect pathogens directly from blood cultures, these tests tend to rely on a selected subset of specific pathogens, meaning that some pathogens will not be detected. Furthermore, many infections do not enter the bloodstream and therefore cannot be detected by non-invasive methods. Thus, there remains a need to assess host responses (such as host gene expression) to classify bacterial and viral infections and to correlate host responses to outcomes such as diagnostic and/or treatment regimens.

Provided herein is a method for estimating a diagnostic score of a test sample using real-time quantitative isothermal amplification. In some embodiments, the method includes obtaining a test sample containing at least one first target nucleic acid, at least one second target nucleic acid, and a reference nucleic acid. Each of the first target nucleic acid, the second target nucleic acid, and the reference nucleic acid includes a mammalian host nucleic acid. The method further includes adding a first aliquot of the test sample to a first reaction vessel for quantitative isothermal amplification of the first target nucleic acid, and adding a second aliquot of the test sample to a second reaction vessel for quantitative isothermal amplification of the second target nucleic acid. Each of the first reaction vessel and the second reaction vessel contains a master mix for isothermal amplification of the first target nucleic acid, the second target nucleic acid, and the reference nucleic acid. The second target nucleic acid has an expected lower abundance in the test sample than the first target nucleic acid. The first aliquot has a first volume. The second aliquot has a second volume that is greater than the first volume. The method further comprises performing a first real-time quantitative isothermal amplification assay in the first reaction vessel by initiating a first reaction in the first reaction vessel, determining a time value to a first threshold for a first target nucleic acid in the first reaction, determining a time value to a first reference threshold for a reference nucleic acid in the first reaction, and estimating a first relative abundance value for the first target nucleic acid in the test sample relative to the reference nucleic acid based on at least the time value to the first threshold and the time value to the first reference threshold. The method further comprises performing a second real-time quantitative isothermal amplification assay in the second reaction vessel by initiating a second reaction in the second reaction vessel, determining a time value to a second threshold for a second target nucleic acid in the second reaction, determining a time value to a second reference threshold for a reference nucleic acid in the second reaction, and estimating a second relative abundance value for the second target nucleic acid in the test sample relative to the reference nucleic acid based on at least the time value to the second threshold and the time value to the second reference threshold. The method further includes estimating a diagnostic score for the test sample based on the first relative abundance value of the first target nucleic acid and the second relative abundance value of the second target nucleic acid.

In another aspect, provided herein is a method of estimating a diagnostic score using real-time quantitative isothermal amplification. In some embodiments, the method includes obtaining a test sample from a mammalian subject. The test sample contains at least one first target nucleic acid, at least one second target nucleic acid, and a reference nucleic acid. Each of the first target nucleic acid, the second target nucleic acid, and the reference nucleic acid has an expected concentration in the test sample that is within the dynamic range of the real-time quantitative isothermal amplification, as validated across a cohort population of interest. The method further includes adding an aliquot of the test sample to at least one reaction vessel containing a master mix for isothermal amplification of the first target nucleic acid, the second target nucleic acid, and the reference nucleic acid; initiating at least one reaction of isothermal amplification in the at least one reaction vessel; determining a time value to a first threshold for the first target nucleic acid in the at least one reaction; determining a time value to a second threshold for the second target nucleic acid in the at least one reaction; determining a time value to a reference threshold for the reference nucleic acid in the at least one reaction; estimating a first relative abundance value of the first target nucleic acid relative to the reference nucleic acid in the test sample based on at least the time value to the first threshold and the time value to the reference threshold; estimating a second relative abundance value of the second target nucleic acid relative to the reference nucleic acid in the test sample based on at least the time value to the second threshold and the time value to the reference threshold; and estimating a diagnostic score for the test sample based on the time value to the first threshold for the first target nucleic acid and the time value to the second threshold for the second target nucleic acid.

In another aspect, provided herein is a method for estimating a diagnostic score by performing real-time quantitative isothermal amplification on a test sample. In some embodiments, the method includes obtaining a first standard curve, a second standard curve, and a reference standard curve. The first standard curve includes a first function relating the starting copy number of the first target nucleic acid to the time to a threshold value. The second standard curve includes a second function relating the starting copy number of the second target nucleic acid to the time to a threshold value. The reference standard curve includes a reference function relating the starting copy number of the reference nucleic acid to the time to a threshold value. The first standard curve, the second standard curve, and the reference standard curve are generated prior to performing real-time quantitative isothermal amplification on the test sample. The method further includes obtaining a test sample from a mammalian subject. The test sample contains a first target nucleic acid, a second target nucleic acid, and a reference nucleic acid. The method further includes adding the test sample to at least one reaction vessel containing a master mix for isothermal amplification of a first target nucleic acid, a second target nucleic acid, and a reference nucleic acid, initiating at least one reaction of isothermal amplification in the at least one reaction vessel, determining a time to a first threshold value for the first target nucleic acid in the at least one reaction, determining a time to a second threshold value for the second target nucleic acid in the at least one reaction, determining a time to a reference threshold value for the reference nucleic acid in the at least one reaction, estimating a first starting copy number of the first target nucleic acid in the test sample based on the time to the first threshold value using a first function of the first standard curve, and estimating a second starting copy number of the second target nucleic acid in the test sample based on the time to the second threshold value using a second function of the second standard curve. estimating a reference starting copy number of the reference nucleic acid in the test sample based on a time to reference threshold value using a reference function provided by the reference standard curve; estimating a first relative abundance value of the first target nucleic acid in the test sample relative to the reference nucleic acid based on a first starting copy number of the first target nucleic acid and the reference starting copy number of the reference nucleic acid; estimating a second relative abundance value of the second target nucleic acid in the test sample relative to the reference nucleic acid based on a second starting copy number of the second target nucleic acid and the reference starting copy number of the reference nucleic acid; estimating a diagnostic score for the test sample based on the first relative abundance value of the first target nucleic acid and the second relative abundance value of the second target nucleic acid; and performing a clinical diagnosis of the disease condition by comparing the diagnostic score of the test sample to a predetermined threshold diagnostic score.

In another aspect, provided herein is an apparatus for estimating a diagnostic score of a test sample using real-time quantitative isothermal amplification. In some embodiments, the apparatus includes a first reaction vessel configured to hold a first aliquot of the test sample, and a second reaction vessel configured to hold a second aliquot of the test sample. The test sample contains a first target nucleic acid, a second target nucleic acid, and a reference nucleic acid. Each of the first target nucleic acid, the second target nucleic acid, and the reference nucleic acid includes a mammalian host nucleic acid. Each of the first reaction vessel and the second reaction vessel contains a master mix for isothermal amplification of the first target nucleic acid, the second target nucleic acid, and the reference nucleic acid. The first aliquot has a first volume, and the second aliquot has a second volume that is greater than the first volume. The second target nucleic acid has a lower expected abundance in the test sample than the first target nucleic acid. The apparatus further includes a computer memory for storing a first threshold fluorescence intensity value and a second threshold fluorescence intensity value. The apparatus further includes a means for initiating a first isothermal amplification reaction in a first reaction vessel that amplifies the first target nucleic acid and the reference nucleic acid. The first isothermal amplification reaction may generate a first fluorescence associated with the first target nucleic acid and a first reference fluorescence associated with the reference nucleic acid. The apparatus further includes a first fluorescence detector and a first reference fluorescence detector optically coupled to the first reaction vessel. The first fluorescence detector is configured to measure the intensity of the first fluorescence as a function of time and to measure a first reference intensity of the first reference fluorescence as a function of time. The apparatus further includes a means for initiating a second isothermal amplification reaction in a second reaction vessel that amplifies the second target nucleic acid and the reference nucleic acid. The second isothermal amplification reaction may generate a second fluorescence associated with the second target nucleic acid and a second reference fluorescence associated with the reference nucleic acid. The apparatus further includes a second fluorescence detector and a second reference fluorescence detector optically coupled to the second reaction vessel. The second fluorescence detector is configured to measure the intensity of the second fluorescence as a function of time and to measure a second reference intensity of the second reference fluorescence as a function of time. The apparatus further includes a computer processor coupled to the first fluorescence detector, the second fluorescence detector, the first reference fluorescence detector, the second reference fluorescence detector and the computer memory. The computer processor determines a first time-to-threshold value for the first target nucleic acid based on the intensity of the first fluorescence as a function of time and the first threshold fluorescence intensity value, determines a first reference time-to-threshold value for the reference nucleic acid based on the intensity of the first reference fluorescence as a function of time and the first threshold fluorescence intensity value, estimates a first relative abundance value for the first target nucleic acid in the test sample relative to the reference nucleic acid based on at least the time-to-first threshold value and the time-to-first reference threshold value, and estimates a first relative abundance value for the first target nucleic acid in the test sample relative to the reference nucleic acid based on the intensity of the second fluorescence as a function of time and the second threshold fluorescence intensity value. determine a time to a second threshold value for the second target nucleic acid based on the value; determine a time to a second reference threshold value for the reference nucleic acid based on the intensity of the second reference fluorescence as a function of time and the second threshold fluorescence intensity value; estimate a second relative abundance value for the second target nucleic acid in the test sample relative to the reference nucleic acid based on at least the time to the second threshold value and the time to the second reference threshold value; and estimate a diagnostic score for the test sample based on the first relative abundance value of the first target nucleic acid and the second relative abundance value of the second target nucleic acid.

1 shows an exemplary plot of fluorescence intensity as a function of time in a real-time quantitative isothermal amplification assay according to some embodiments. 1 shows an exemplary plot of fluorescence intensity as a function of time in a real-time quantitative isothermal amplification assay of eight samples with different concentrations of target nucleic acid, according to some embodiments. 3 shows a scatter plot of data points representing time to threshold values versus the logarithm of copy number obtained from the fluorescence curves shown in FIG. 2 and the line obtained from linear regression of the data points, according to some embodiments. 1 shows a simplified flowchart illustrating a method for estimating a diagnostic score by performing real-time quantitative isothermal amplification on a test sample, according to some embodiments. 1 shows a simplified flowchart illustrating a method for estimating a diagnostic score by performing real-time quantitative isothermal amplification of a test sample, according to some other embodiments. 1 shows a simplified flowchart illustrating a method for estimating a diagnostic score by performing real-time quantitative isothermal amplification on a test sample, according to some embodiments. FIG. 1 shows a schematic block diagram of an apparatus for estimating relative abundance values using a real-time quantitative isothermal amplification assay, according to some embodiments. Graph showing correlation plot with Pearson's coefficient for the relative abundance of target nucleic acid (IFI27) to reference nucleic acid (YWHAB) determined using a quantitative real-time isothermal amplification assay compared to a gold standard assay (NanoString nCounter). Values are plotted as _Log2 (Fold Abundance) for both assays and assigned according to diagnosis: circles represent viral infections and squares represent bacterial sepsis. Triangles represent healthy controls. Lines represent curve fits by standard linear regression analysis. 1 shows a correlation plot with Pearson coefficients associated with diagnostic scores determined by an isothermal amplification assay and the gold standard NanoString nCounter, according to some embodiments. 1 shows HostDx-Fever score distribution based on qLAMP assay, according to some embodiments. 1 shows the HostDx-Fever score distribution based on NanoString nCounter according to some embodiments. 1 shows an example in accordance with some embodiments in which the cutoff value is defined as the y-intercept of a linear regression fit to a standard curve titration. 1 shows experimental results of a qLAMP assay using multi-volume reaction vessels, according to some embodiments.

Before describing the invention, it is to be understood that the invention is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

The present disclosure provides methods, devices, and kits for determining the relative abundance of a target nucleic acid in a test sample. While qPCR is well known in the art, isothermal amplification techniques have failed to further advance methods for quantifying target nucleic acids in clinically relevant samples. For example, qLAMP methods for quantifying bacterial or viral nucleic acids may not be reliable below 1,000 copies (see Nixon et al., (2014), Bimolecular Detection and Quantitation 2:4-10). What is needed, therefore, is a method for quantifying the presence of a target mammalian mRNA in a sample, independent of the starting copy number of the target nucleic acid in the sample. Provided herein are methods, devices, and kits for determining the relative abundance of a target nucleic acid relative to a reference nucleic acid in a test sample that is not based on the starting copy number and does not require absolute quantification of the target nucleic acid or reference nucleic acid in the sample. These methods, devices, and kits utilize real-time quantitative isothermal amplification to amplify the target nucleic acid and reference nucleic acid in the test sample. In some embodiments, the target nucleic acid is a mammalian host nucleic acid (e.g., mRNA) expressed by the host in response to bacterial or viral infection. Relative quantification is sufficient to allow calculation of certain diagnostic algorithms that rely on multiple mammalian RNA markers (see, e.g., Published Patent Applications WO2016/145426, WO2017/066641, WO2018/004806, and WO2017/214061) stably across samples without the need to run a standard curve for each target in the same assay, or even without the use of a standard curve.

In some embodiments, quantitative real-time isothermal amplification includes strand displacement amplification (SDA), transcription-mediated amplification (TMA), nucleic acid sequence-based amplification (NASBA), recombinase polymerase amplification (RPA), rolling circle amplification (RCA), branched amplification, helicase-dependent isothermal DNA amplification (HDA), nicking enzyme amplification reaction (NEAR), and loop-mediated isothermal amplification (LAMP) (e.g., Notomi et al., (2000) Nucleic Acids Research, 28(12)E63, incorporated herein by reference).

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described. All publications and accession numbers mentioned herein are incorporated by reference to disclose and describe the methods and/or materials in connection with which the publications are cited.

In general, the nomenclature used herein and the laboratory procedures in cell culture, molecular biology, organic chemistry, and nucleic acid chemistry and hybridization described below are those well known and commonly used in the art. Standard techniques are used for nucleic acid and peptide synthesis. These techniques and procedures are generally carried out according to conventional methods in the art and various general references provided throughout this document (see generally Sambrook et al. Molecular Cloning: A Laboratory Manual, 2d ed. (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., incorporated herein by reference).

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this subject matter belongs. All patents, patent applications, published applications and publications, GENBANK sequences, websites, and other publications mentioned throughout this disclosure are incorporated by reference in their entirety unless otherwise indicated. In the event of a plurality of definitions for terms herein, the definitions in this section prevail. When referring to a URL, or other such identifier or address, it is understood that such identifiers may change and specific information on the Internet may come and go, but that equivalent information is known and readily accessible, such as by searching the Internet and/or suitable databases. Reference thereto evidences the availability and public dissemination of such information.

As used herein, the terms "a," "an," and "the" are defined to mean one or more, and include the plural unless the context is inappropriate. In this application, the use of the singular includes the plural unless otherwise indicated.

Ranges can be expressed herein as "about" one particular value and/or to "about" another particular value. As used herein, the term "about" means approximately, in the region of, roughly, or around. When the term "about" is used in conjunction with a numerical range, the term modifies that range by extending the boundaries above and below the stated numerical values. In general, the term "about" is used herein to modify numerical values that are a 10% variance above and below the stated value. When such ranges are expressed, another embodiment includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by using the antecedent "about," it is understood that the specified value forms another embodiment. It is further understood that the endpoints of each range are included in the range.

As used herein, the word "or" means any one member of a particular list and also includes any combination of members of that list.

As used herein, the term "including," as well as other forms thereof (e.g., "include," "includes," and "included"), are not limiting.

As used herein, the term "relative abundance value" refers to a measurement of a target nucleic acid in a test sample. In some embodiments, the relative abundance value is estimated by performing isothermal amplification of the target nucleic acid and housekeeping genes in the test sample such that differences in gene expression can be compared between different samples (e.g., samples obtained from different subjects or samples obtained from a single subject at different times (e.g., pre-treatment and post-treatment)).

As used herein, the term "target nucleic acid" refers to a mammalian RNA sequence expressed from a mammalian host gene, for example, in response to pathogenic (e.g., bacterial or viral) activity. Mammalian RNA can include humans and non-human mammals such as horses, cats, dogs, pigs and sheep. In a preferred embodiment, the target nucleic acid is a mammalian mRNA. In some embodiments, the target nucleic acid encompasses a splice junction within the mammalian host mRNA.

As used herein, the term "reference nucleic acid" or "endogenous control" refers to a nucleic acid present in a test sample that is used to normalize the amount of a target nucleic acid. A reference nucleic acid may include a naturally occurring nucleic acid that is normally found in a test sample (such as a housekeeping gene mRNA) or may include a known amount of input material spiked into the test sample to normalize the amount of the target nucleic acid.

As used herein, the term "test sample" refers to a biological sample obtained from a mammal. In some cases, the biological sample is a clinical sample obtained during a clinical procedure or obtained by a physician or physician assistant, such as a cervical or vaginal swab, a nasal swab, a blood or blood component sample (such as plasma, serum or PMBC). In some embodiments, the biological sample includes an excreted biological sample, such as a mucus, stool or urine sample. In some embodiments, the test sample is a self-collected specimen, such as a nasal swab, buccal swab, finger stick blood, etc.

The term "isothermal amplification" refers to a process in which a target nucleic acid is amplified using a single constant amplification temperature (e.g., about 30°C to about 95°C). Unlike standard PCR, isothermal amplification reactions do not involve multiple cycles of denaturation, hybridization, and extension of annealed oligonucleotides to form a population of amplified target nucleic acid molecules (i.e., amplicons). There are various types of isothermal applications known in the art, including, but not limited to, loop-mediated isothermal amplification (LAMP), nucleic acid sequence-based amplification (NASBA), recombinase polymerase amplification (RPA), rolling circle amplification (RCA), nicking enzyme amplification reaction (NEAR) and helicase-dependent amplification (HDA).

As used herein, the term "real-time quantitative isothermal amplification" refers to a process in which a target nucleic acid is amplified at a constant temperature and the rate of amplification of the target nucleic acid is monitored by fluorescence, turbidity, or similar means (such as NEAR or LAMP). In some embodiments, RNA (such as mRNA) is isolated from a biological sample and used as a template to synthesize cDNA. Reverse transcription is a well-known method for converting mRNA to cDNA. The cDNA molecules are amplified under isothermal amplification conditions so that the amplified target nucleic acid product can be detected and quantified. As used herein, "amplification rate" is the rate at which a target nucleic acid amplicon is produced.

As used herein, "polymerase" refers to one or more enzymes used with strand displacement activity to carry out template-directed synthesis of polynucleotides, e.g., DNA and/or RNA. The term encompasses both full-length polypeptides and domains with polymerase activity. Further examples of commercially available polymerase enzymes include, but are not limited to, Klenow fragment (New England Biolabs® Inc.), Taq DNA polymerase (QIAGEN), 9°N™ DNA polymerase (New England Biolabs® Inc.), Deep Vent™ DNA polymerase (New England Biolabs® Inc.), Manta DNA polymerase (Enzymatics®), Bst DNA polymerase (New England Biolabs® Inc.), and phi29 DNA polymerase (New England Biolabs® Inc.). Polymerases such as Bst2.0, Bst3.0 and Bst2.0 WarmStart® DNA polymerase (all commercially available from New England Biolabs® Inc.) are suitable for LAMP. LAMP can be applied to RNA when reverse transcriptase (RTase) is used in conjunction with the DNA polymerase.

Polymerases include both DNA-dependent polymerases and RNA-dependent polymerases, such as reverse transcriptase. At least five families of DNA-dependent DNA polymerases are known, but most are classified into families A, B, and C. Other types of DNA polymerases include phage polymerases. Similarly, RNA polymerases typically include eukaryotic RNA polymerases I, II, and III, as well as bacterial RNA polymerases, and phage and viral polymerases. RNA polymerases can be DNA-dependent and RNA-dependent.

As used herein, the term "standard curve" is given its clear and ordinary meaning in the art. Typically, a standard curve is derived from a set of samples of different concentrations of the target nucleic acid, e.g., by serial dilution of the template. The time-to-threshold values are plotted versus the logarithm (e.g., base 10) of the concentration. A least-squares fit is used as the standard curve.

As used herein, the term "master mix for isothermal amplification" refers to multiple components, such as dNTPs, salts (e.g., magnesium), and buffers, required to perform an isothermal amplification assay. In some cases, the master mix for isothermal amplification is a real-time quantitative isothermal amplification master mix. In some embodiments, the master mix for isothermal amplification contains all components required to perform an isothermal amplification reaction, except for the primers, probes, and nucleic acid templates to be amplified.

The term "fluorescent label" or "fluorophore" refers to a compound having a fluorescence emission maximum between about 400 and about 900 nm. These compounds, with their emission maxima (in nm) in parentheses, include: Syto-9, Syto-82, Cy2™ (506), GFP (red shifted) (507), YO-PRO™-1 (509), YOYO™-1 (509), Calcein (517), FITC (518), FluorX™ (519), Alexa™ (520), Rhodamine 110 (520), 5-FAM (522), Oregon Green™ 500 (522), Oregon Green™ 488 (524), Ribo Green™ (525), Rhodamine Green™ (527), Rhodamine 110 (528), 5-FAM (529), Oregon Green™ 500 (529), Oregon Green™ 488 (530), Ribo Green™ (531), Rhodamine Green™ (532), Rhodamine 110 (533), Rhodamine 110 (534), Rhodamine 110 (535), Rhodamine 110 (536), Rhodamine 110 (537), Rhodamine 110 (538), Rhodamine 110 (539), Rhodamine 110 (540), Rhodamine 110 (541), Rhodamine 110 (542), Rhodamine 110 (543), Rhodamine 110 (544), Rhodamine 110 (545), Rhodamine 110 (546), Rhodamine 110 (547), Rhodamine 110 (548), Rhodamine 110 (549), Rhodamine 110 (550), Rhodamine 110 (551), Rhodamine 110 (552), Rhodamine 110 (5 123 (529), MagnesiumGreen™ (531), CalciumGreen™ (533), TO-PRO™-1 (533), TOTO™-1 (533), JOE (548), BODIPY™ 530/550 (550), Di1 (565), BODIPY™ 558/568 (568), BODIPY™ 564/570 (570), Cy3™ (570), Alexa™ 546 (570), TRITC (572), MagnesiumOrange™ (575), Phycoerythrin R&B (575), Rhodamine Phalloidin (575), CalciumOrange™ (576), Pyronin Y (580), Rhodamine B (580), TAMRA (582), RhodamineRed™ (590), Cy3.5™ (596), ROX (608), Calcium Crimson™ (615), Alexa™ 594 (615), TexasRed™ (615), Nile Red (628), YO-PRO™-3 (631), YOYO™-3 (631), R-phycocyanin (642), C-Phycocyanin (648), TO-PRO™-3 (660), TOTO®-3 (660), DiD DilC(5) (665), Cy5™ (670), Thiadicarbocyanine (671) and Cy5.5 (694). Additional fluorophores are disclosed in PCT Patent Publication No. WO 03/023357 and U.S. Patent No. 7,671,218. Examples of these and other suitable fluorescent dye classes are described by Haugland et al. , Handbook of Fluorescent Probes and Research Chemicals, Sixth Ed. , Molecular Probes, Eugene, Ore. (1996); U.S. Patent Nos. 3,194,805, 3,128,179, 5,187,288, 5,188,934, 5,227,487, 5,248,782, 5,304,645, 5,433,896, 5,442,045, 5,556,959, 5,583,236, 5,808,044, 5,852,191, 5,986,086, 6,020,481, 6,162,931, 6,180,295 and 6,221,604, European Patent No. 1408366; Smith et al., J. Chem. Soc. Perkin Trans. 2:1195-1204 (1993); Whitaker, et al. , Anal. Biochem. 207:267-279 (1992); Krasovskii and Bolotin, Organic Luminescent Materials, VCH Publishers, NY. (1988); Zolliger, Color Chemistry, 2nd Edition, VCH Publishers, NY. (1991); Hirschberg et al. , Biochemistry 37:10381-10385 (1998); Fieser and Fieser, REAGENTS FOR ORGANIC SYNTHESIS, Volumes 1 to 17, Wiley, US (1995); and Geiger et al., Nature 359:859-861 (1992).

There is extensive guidance in the art for selecting quencher and fluorophore pairs and their attachment to oligonucleotides (e.g., Haugland, 1996, U.S. Pat. Nos. 3,996,345 and 4,351,760). Exemplary quenchers are described in U.S. Pat. No. 6,727,356, which is incorporated herein by reference. Other quenchers include, but are not limited to, bis-azo quenchers (U.S. Pat. No. 6,790,945) and dyes from Biosearch Technologies, Inc. (offered as Black Hole™ Quenchers: BH-1, BH-2 and BH-3 quenchers), Dabcyl, TAMRA and carboxytetramethylrhodamine.

Any suitable fluorophore can be used to fluorescently label the primers, probes or nucleotides incorporated during the isothermal amplification reaction. In some embodiments, the fluorescent label is an intercalating agent, such as, but not limited to, Syto-9, Syto-82, SYBR®, Hoechst 33258 or DAPI.

As used herein, the term "time to threshold" refers to the time elapsed from the moment isothermal amplification is initiated to the moment that the fluorescence intensity, which represents the concentration of the target nucleic acid, reaches a predetermined threshold. In a plot of fluorescence intensity versus time since isothermal amplification was initiated (e.g., as shown in FIG. 1), the time to threshold is the x-axis crossing point where the fluorescence curve intersects the horizontal line representing the threshold.

As used herein, the term "copy number" refers to the number of target or reference nucleic acid molecules present in the original test sample (i.e., prior to real-time quantitative isothermal amplification).

As used herein, the term "N-fold serial dilution" refers to the stepwise dilution of a first component of a solution (such as a reference nucleic acid) with a certain dilution factor (1:5, 1:10, 1:20 or 1:50) using a suitable buffer (such as saline, water, etc.). For example, a 10-fold serial dilution requires serial dilution of a first component (such as a reference nucleic acid present at a concentration of 100 mg/ml) with a dilution factor of 10 (such as 1:10) to form a first serial dilution of the reference nucleic acid present at a concentration of 10 mg/ml, followed by a second 10-fold dilution of the first serial dilution (e.g., to form a second serial dilution of the reference nucleic acid present at a concentration of 1 mg/ml), etc.

As used herein, the term "linear regression" refers to a statistical linear approach to modeling the relationship between a dependent variable and one or more independent variables. In linear regression, the relationship is modeled using linear predictor functions where unknown model parameters are estimated from the data. Such models are called linear models. Typically, for a single independent variable, (x,y) data points are plotted in graphical form as a scatter plot, where x is the independent variable and y is the dependent variable. Linear regression aims to obtain a "best fit line" that represents the relationship between the dependent and independent variables. Linear regression models are often fitted using a least squares approach, but may also be fitted in other ways, such as minimizing a "cost function" in other norms (e.g., L1-norm or L2-norm penalization).

As used herein, the term "linear dynamic range" of a quantitative isothermal amplification assay refers to the range of input concentrations of target nucleic acid over which a plot of time to threshold versus logarithm of concentration (e.g., base 2 or base 10) is linear.

As used herein, the term "dynamic range" refers to the range (maximum to minimum) of sample concentrations or input amounts that a given assay (e.g., LAMP) can detect.

As used herein, the term "diagnostic score" refers to an integrated score for classifying or diagnosing a disease state. A diagnostic score combines the expression levels of several biomarkers identified as associated with a disease state using a specific algorithm into a single score to which a clinically relevant threshold can be applied. Exemplary algorithms are described in Sections J, K, and L below.

As used herein, the term "population study" refers to the study of a clinically representative population to establish statistics of the expression levels of one or more biomarkers. As used herein, the term "clinically representative population" refers to a group of individuals large enough to establish statistical significance. For example, in a population study, biomarkers can be measured in patients with and without a particular disease and a Student's t-test, Welch's t-test, Mann-Whitney U test, or analysis of variance (ANOVA), F-test, etc., can be used to determine whether the biomarkers are differentially expressed. Statistical significance can be set at, for example, p<0.05.

As used herein, the term "machine learning model" refers to an algorithm or statistical model that a computer system uses to perform a task that relies on patterns and inferences from a set of test data. A particular algorithm that integrates several biomarkers into a diagnostic score may be a machine learning model. Exemplary machine learning models include linear regression, logistic regression, decision trees, support vector machines (SVMs), random forests, K-means, artificial neural networks (ANNs), and the like.

As used herein, the term "hot start" refers to a variation of the amplification process that prevents non-specific amplification of nucleic acids by inactivating the polymerase used for amplification. Hot start reduces primer-dimer formation and non-specific annealing of primers to non-target nucleic acids that are subsequently extended during amplification. There are various approaches to hot start (see, for example, Paul et al., (2010) Methods Mol Biol., 630:301-18). For example, the polymerase can be inactivated by using antibodies, aptamers or chemical modifications such that the polymerase (which may be moderately active at room temperature and/or on ice) is prevented from functioning at suboptimal temperatures. In the hot start process, an initial activation step (such as 95°C) is performed to activate the polymerase. This initial heat activation step also inactivates antibodies linked to the polymerase or removes chemical modifications (such as lysine modifications) from the polymerase. Once these components are inactivated, the polymerase is able to amplify the target nucleic acid present in the test sample. In some embodiments, the methods disclosed herein include a hot-start mechanism, such as a chemical modification to an antibody, aptamer, or polymerase used in a real-time quantitative isothermal amplification assay.

"Primer" refers to a polynucleotide sequence that hybridizes to a complementary sequence on a target nucleic acid and serves as a starting point for nucleic acid synthesis. Primers can be of various lengths and are often less than 80 nucleotides in length, e.g., 20-70 nucleotides in length. In one embodiment, the full-length target-specific primers of the present application can comprise 30-65 nucleotides in length, e.g., 30, 35, 40, 45, 50, 55, 60 or 65 nucleotides in length. In some embodiments, a multiplex amplification reaction mixture can comprise one or more full-length target-specific primers that are 20-65 nucleotides in length. Target-specific primers of different lengths can be used in a multiplex amplification reaction such that one or more of the multiplex full-length target-specific primers comprise a different nucleotide length compared to the remainder of the multiplex full-length target-specific primer pair (such as a multiplex amplification reaction comprising a first full-length target-specific primer having a nucleotide length of 45 nucleotides and a second full-length target-specific primer having a nucleotide length of 58 nucleotides). The length and sequence of primers for use in isothermal amplification can be designed based on principles known to those of skill in the art. For example, Innis et al. , PCR Protocols: A Guide to Methods and Applications (Innis et al., eds, 1990). There are various tools for primer design and evaluation (such as NCBI-Blast software). In this application, primers with highly degenerate sequences can be avoided to reduce the possibility of mishybridization during the isothermal amplification reaction. Primers can be prepared from DNA, RNA, or chimeras of DNA and RNA portions. In some cases, primers can contain one or more modified nucleosides (such as 2-amino-deoxyadenosine) or non-natural nucleotide bases (such as uracil in DNA primers). In some cases, primers can contain fluorescent labels, such as FRET donor and FRET acceptor moieties.

As used herein, the term "probe" refers to an oligonucleotide capable of hybridizing to another oligonucleotide of interest, whether naturally occurring, synthetically, recombinantly, or produced by amplification. Probes can be single-stranded or double-stranded. Probes are useful for the detection, identification, and isolation of specific gene sequences. In one embodiment, it is contemplated that any probe used in the present invention is labeled such that the label is detectable in any detection system, including fluorescent, radioactive, and luminescent systems. It is not intended that the present invention be limited to a particular detection system or label.

As used herein, the term "amplicon" retains its normal and customary usage in the art. An amplicon is an amplification product generated from a DNA or cDNA template.

As used herein, the term "relative quantification" refers to a comparison between the expression level of a target nucleic acid in a single sample and the expression level of a reference nucleic acid, or a comparison between the expression levels of the same target nucleic acid in different samples. Relative quantification is in contrast to "absolute quantification," which involves determining the absolute amount of a target nucleic acid in a sample.

A. Test Samples In some embodiments, the methods, devices and associated kits described herein utilize a test sample. In some embodiments, the test sample is obtained from a clinical sample (e.g., nasal swab, biopsy or blood draw) taken for diagnostic evaluation to identify a disease or condition. In some embodiments, the test sample is obtained by a physician or veterinarian. In some embodiments, the test sample is a self-collected specimen, such as a nasal swab, buccal swab, etc. In some embodiments, the test sample is obtained by a medical device (e.g., a leukapheresis device). Any suitable test sample may be used to practice the present invention.

In some embodiments, the sample is obtained from a mammal, such as, but not limited to, a human. In some embodiments, the sample is obtained from a non-human mammal, such as, but not limited to, a chimpanzee, cat, dog, pig, sheep, or cow. In some embodiments, the test sample is obtained from a healthy or diseased mammal. In some embodiments, the sample is obtained from a mammalian subject diagnosed with or suspected of having a viral or bacterial infection. In some embodiments, the mammalian subject is suspected of having a bacterial infection (e.g., Streptococcus). In some embodiments, the mammalian subject is suspected of having a viral infection (e.g., HIV).

In some embodiments, the test sample obtained from the host includes a bodily fluid such as urine, saliva, blood or blood components (such as, but not limited to, serum, plasma, and peripheral blood mononuclear cells (PMBCs)). Any suitable bodily fluid may be used to practice the invention.

B. Target Nucleic Acids In some embodiments, the methods, devices, and associated kits described herein utilize target nucleic acids. In some embodiments, the target nucleic acid is a mammalian nucleic acid. In some embodiments, the target nucleic acid is a host nucleic acid (e.g., produced by a cell of a host or detected in a sample obtained from a source of a test sample). In some embodiments, the target nucleic acid is a mammalian host nucleic acid (such as mammalian DNA or RNA). In some embodiments, the target nucleic acid is a mammalian host RNA. In a preferred embodiment, the target nucleic acid is a mammalian host mRNA. In one embodiment, the target nucleic acid includes a splice junction within the mammalian host mRNA. In one embodiment, the target nucleic acid includes a nucleic acid of pathogen origin that is detected simultaneously with the mammalian host mRNA.

In some embodiments, the target nucleic acid is present in a sample obtained from a mammalian host and the target nucleic acid is isolated from the host sample (e.g., RNA extraction). In some embodiments, the target nucleic acid is present in a sample obtained from a mammalian host (e.g., whole blood) and the target nucleic acid is isolated from the host sample (e.g., QIAamp RNA Blood Mini Kit, Qiagen, Catalog No.: 52304).

In some embodiments, the mammalian host target nucleic acid is more abundant in the test sample compared to the reference nucleic acid. In some embodiments, the mammalian host target nucleic acid is less abundant in the test sample compared to the reference nucleic acid. Any suitable mammalian host nucleic acid may be used to practice the invention.

C. Reference Nucleic Acid In some embodiments, the methods, devices and associated kits described herein utilize a reference nucleic acid. In some embodiments, the reference nucleic acid is a mammalian nucleic acid. In some embodiments, the reference nucleic acid is a host nucleic acid (e.g., produced by a cell of a host or detected in a sample obtained from a source of a test sample). In some embodiments, the reference nucleic acid is a mammalian host nucleic acid (such as a mammalian DNA or RNA). In some embodiments, the reference nucleic acid is a mammalian host RNA. In a preferred embodiment, the reference nucleic acid is a mammalian host mRNA. In some embodiments, the mammalian host nucleic acid is more abundant in the test sample compared to the target nucleic acid. In some embodiments, the mammalian host nucleic acid is less abundant in the test sample compared to the target nucleic acid. In a preferred embodiment, the reference nucleic acid and the target nucleic acid are expressed by different genes. In yet another embodiment, the reference nucleic acid and the target nucleic acid are expressed by different genes from the same mammalian organism (e.g., human or mouse). Any suitable reference nucleic acid may be used to practice the invention.

Housekeeping Genes In some embodiments, the reference nucleic acid is a housekeeping gene or its product, such as a corresponding mRNA transcript. In some embodiments, the reference nucleic acid comprises an mRNA transcript that is a pre-mRNA molecule, a 5' capped mRNA molecule, a 3' adenylated mRNA molecule, or a mature mRNA molecule. In a preferred embodiment, the reference nucleic acid is a mature mRNA molecule obtained from a mammalian host that is also the source of the test sample.

In some embodiments, the reference nucleic acid is a mammalian housekeeping gene or its gene product (e.g., mRNA). In some embodiments, the housekeeping gene is a gene that is constitutively expressed (i.e., continuously transcribed) by one or more cell populations in a mammalian host. A constitutively expressed gene may be contrasted with a facultative gene, which refers to a gene that is transcribed only when necessary. Exemplary housekeeping genes that are suitable for use with the present invention include actin, GAPDH, and ubiquitin. Any suitable housekeeping gene may be used to practice the present invention.

In some embodiments, a housekeeping gene or its product is expressed at a relatively constant rate by the host's cells, such that the rate of expression of the housekeeping gene can be used as a reference point for the expression of other host genes or their gene products.

In some embodiments, the reference nucleic acid is a human housekeeping gene. Exemplary human housekeeping genes suitable for use with the present invention include, but are not limited to, KPNA6, RREB1, YWHAB, chromosome 1 open reading frame 43 (C1orf43), charged multivesicular body protein 2A (CHMP2A), ER membrane protein complex subunit 7 (EMC7), glucose-6-phosphate isomerase (GPI), proteasome subunit, beta type 2 (PSMB2), proteasome subunit, beta type 4 (PSMB4), member RAS oncogene family (RAB7A), receptor accessory protein 5 (REEP5), small nuclear ribonucleoprotein D3 (SNRPD3), valosin-containing protein (VCP), and vacuolar protein sorting 29 homolog (VPS29). In some embodiments, any of the housekeeping genes provided at http://www/tau/ac/il-elieis/HKG/ can be used (see Eisenberg and Levanon., Trends Genet. (2013), 10:569-74).

In some embodiments, the reference nucleic acid is a porcine housekeeping gene. Exemplary porcine housekeeping genes suitable for use with the present invention include, but are not limited to, ACTB, B2M, GAPDH, HMBS, SDHA, HPRT1, TBP, YWHAZ, and RPL32.

In some embodiments, the reference nucleic acid is a bovine housekeeping gene. Exemplary bovine housekeeping genes suitable for use with the present invention include, but are not limited to, ACTB, GAPDH, HMBS, SF3A1 HPRT1, H2A, and SDHA.

In some embodiments, the reference nucleic acid is an equine housekeeping gene. Exemplary bovine housekeeping genes suitable for use with the present invention include, but are not limited to, ACTB, GAPDH, TOP2B, KRT8, and RPS9.

D. Test sample processing 1. Reaction vessel In some embodiments, the target nucleic acid and reference nucleic acid from the test sample are provided in a first reaction vessel.As used herein, "reaction vessel" refers to a system that can carry out the present invention, including but not limited to test tubes, microcentrifuge tubes, wells, chambers, microwells (e.g., 96, 384, and 1536 well assay plates), capillary tubes, microfluidic devices, or test sites on or in suitable surfaces (including but not limited to glass, plastic, silicon, metal oxide, beads, and silanized (e.g., alkoxysilane) surfaces).Any suitable reaction vessel may be used to carry out the present invention.

In some embodiments, the reaction vessel contains less than 1000 μL of liquid during quantitative isothermal amplification. In other embodiments, the reaction vessel contains about 15 μL to about 750 μL of liquid during quantitative isothermal amplification.

In another embodiment, the target nucleic acid from the test sample is contained in a first reaction vessel and the reference nucleic acid from the test sample is contained in a second reaction vessel. The reaction vessels can be of any useful dimensions (width, length, height, etc.) and can be constructed of any suitable material. Preferably, the reaction vessel(s) of the present application are in the microliter or nanoliter scale.

In some embodiments, multiple reaction vessels are used for multiple target nucleic acids, with each reaction vessel being used for the isothermal amplification of a respective target nucleic acid.

In some embodiments, the reaction vessel can further include a capture region for isolating a target nucleic acid or a reference nucleic acid from a test sample before, after, or during quantitative isothermal amplification. In one embodiment, the reaction vessel can include a capture region for isolating a target nucleic acid and/or a reference nucleic acid from a test sample after quantitative isothermal amplification. In some embodiments, the capture region can include a filter, matrix, polymer, gel, and membrane, including any of those described herein (e.g., silica membrane, glass fiber membrane, cellulose membrane, nitrocellulose membrane, polysulfone membrane, nylon membrane, polyvinylidene fluoride membrane, vinyl copolymer membrane or ion exchange membrane, fiber (e.g., glass fiber), or particle (silica particle, bead, affinity resin or ion exchange resin, etc.).

Any suitable material may be used as the reaction vessel. The material used to form the reaction vessel is selected for the physical and chemical properties desired for proper functioning of the reaction vessel. Suitable materials include silicone polymers (e.g., polydimethylsiloxane and epoxy polymers), polyimides (e.g., commercially available Kapton® (poly(4,4'-oxydiphenylene-pyromellitimide, DuPont, Wilmington, Delaware) and Upilex™ (poly(biphenyltetracarboxylic dianhydride), Ube Industries, Ltd., Japan)), polycarbonates, polyesters, polyamides, polyethers, polyurethanes, polyfluorocarbons, fluorinated polymers (e.g., polyvinylidene fluoride, polyvinylidene fluoride, polytetrafluoroethylene, polychlorotrifluoroethylene, perfluoroalkoxy polymers, fluorinated ethylene-propylene, polyethylene tetrafluoroethylene, polyethylene chlorotrifluoroethylene, perfluoropolyethylene, perfluorosulfonic acid, perfluoropolyimides, FFPM/FFKM (perfluorinated elastomers [perfluoroelastomers]), FPM/FKM (fluorocarbons), Polymeric materials such as acrylic acid polymers such as acrylic acid [chlorotrifluoroethylene vinylidene fluoride], and copolymers thereof, polyether ether ketone (PEEK), polystyrene, poly(acrylonitrile-butadiene-styrene) (ABS), acrylates and polymethyl methacrylates, and other substituted and unsubstituted polyolefins (e.g., cycloolefin polymers, polypropylene, polybutylene, polyethylene (PE, e.g., crosslinked PE, high density PE, medium density PE, linear low density PE, low density PE or ultra-high molecular weight PE), polymethylpentene, polybutene-1, polyisobutylene, ethylene propylene rubber, ethylene propylene diene monomer (M class) rubber), and copolymers thereof (e.g., cycloolefin copolymers); ceramics such as aluminum oxide, silicon oxide, zirconium oxide; semiconductors such as silicon, gallium arsenide; glass; metals; and coated combinations, composites and laminates thereof.

2. Hot Start Isothermal Amplification Timing Mechanism In some embodiments, the methods, devices, and kits provided herein utilize various mechanisms to ensure amplification of target nucleic acids and reference nucleic acids can be reliably compared (e.g., to each other and across various non-sequential experimental replicates). In some cases, the methods and devices include a "hot start" mechanism that reduces or inhibits the generation of non-specific (e.g., spurious) amplification products in the test sample. A hot start can ensure that each reaction begins at the same time. Various techniques for performing hot start amplification are known in the art (see, e.g., Paul et al., (2010) Methods Mol Biol., 630:301-18). Any suitable hot start mechanism may be used to practice the present invention.

In some embodiments, the "hot start" mechanism includes one or more antibodies that inactivate a polymerase (e.g., an RNA polymerase or a DNA polymerase) in a quantitative isothermal amplification assay.

In some embodiments, the "hot start" mechanism includes one or more aptamers that inactivate the polymerase of a quantitative isothermal amplification assay (see, e.g., WarmStart LAMP Kit, New England Biolabs Catalog No. E1700S; and WarmStart RTx Reverse Transcriptase, New England Biolabs Catalog No. M0380S). Nucleic acid-based aptamers suitable for use with polymerases include those described in U.S. Patent Nos. 5,475,096, 5,670,637, 5,696,249, 5,874,557, and 5,693,502.

In some embodiments, the "hot start" mechanism comprises one or more chemical modifications that inactivate the polymerase of the quantitative isothermal amplification assay. In some embodiments, these one or more chemical modifications comprise a lysine modification in which one or more lysine residues present in the polymerase are chemically modified such that the polymerase is inactive until the amplification reaction reaches a temperature above 90°C.

In the hot start mechanism, heat is used to activate the polymerase. This initial heat activation may also inactivate antibodies linked to the polymerase or remove chemical modifications (e.g., lysine modifications) from the polymerase. Once these components are inactivated, the polymerase is able to amplify the target nucleic acid present in the test sample.

Co-amplification In some embodiments, the methods, devices and kits provided herein utilize a co-amplification step to ensure that the amplification of the target nucleic acid and the reference nucleic acid can be reliably compared (e.g., to each other and across various non-sequential experimental replicates). In some embodiments, the amplification of the target nucleic acid in the test sample (e.g., in a first reaction vessel) occurs simultaneously with the amplification of the reference nucleic acid in the test sample (in a second reaction vessel). In some embodiments, the co-amplification step includes amplifying the target nucleic acid and the reference nucleic acid at the same time (e.g., identical start and stop amplification reaction times) in the same reaction vessel. In some embodiments, the co-amplification may include a "hot start" mechanism such that the amplification of the target nucleic acid and the reference nucleic acid (in the same or different reaction vessels) does not begin until each of the target nucleic acid and the reference nucleic acid reaches the same designated temperature (e.g., 65°C), at which point the amplification reactions begin simultaneously.

Non-simultaneous amplification In some embodiments, the methods, devices and kits provided herein utilize a non-simultaneous amplification step of the target nucleic acid and the reference nucleic acid using a time indicator to initiate the amplification reaction so that two or more reactions can be reliably compared (e.g., with each other and across various non-sequential experimental repetitions). In some embodiments, the amplification of the target nucleic acid in the test sample (e.g., in a first reaction vessel) occurs non-concurrently compared to the amplification of the reference nucleic acid in the test sample (e.g., in a second reaction vessel). In some embodiments, non-simultaneous amplification includes amplifying the target nucleic acid and the reference nucleic acid in the same reaction vessel, for example, by providing a primer and/or probe complementary to the target nucleic acid at time 1 and a primer and/or probe complementary to the reference nucleic acid at a later time (e.g., time 2). To obtain a relative abundance value of the target nucleic acid in the test sample using a non-simultaneous amplification approach, it is important to determine the length of the amplification reaction for the amplification of the target nucleic acid. Once the length of the amplification reaction is known, it can be used as a basis for carrying out the amplification of the reference nucleic acid. In this way, each of the target nucleic acid and the reference nucleic acid is amplified for the same period and under the same conditions so that the output of the amplification reaction can be reliably compared to each other.

E. Isothermal Amplification Described herein are methods, devices, and associated kits for isothermal amplification of target nucleic acids. Isothermal amplification assays do not include the cyclic heating and cooling steps required in PCR. As such, isothermal amplification assays do not require expensive equipment such as thermocyclers to perform isothermal amplification (see, e.g., Gill and Ghaemi, (2008) Nucleos. Nucleot. Nucl., 27:224-243; Kim and Easley (2011), Bioanalysis, 3:227-239; and Yan et al., Mol. Biosyst., 10:970-1003). In some embodiments, isothermal amplification comprises a real-time isothermal amplification assay. In some embodiments, the isothermal amplification assay is a real-time quantitative isothermal amplification assay. Real-time isothermal amplification, also called quantitative isothermal amplification, is often used to measure the amount of amplification product in real time. Quantitative isothermal amplification typically involves the use of a labeled probe (e.g., a fluorophore-containing probe or fluorescent dye) along with a series of standards in the amplification reaction that allows for the quantification of the starting amount of target nucleic acid in a sample.

In some embodiments, isothermal amplification includes reverse transcriptase and a strand-displacing isothermal enzyme, such as, but not limited to, Bst polymerase. Reverse transcriptase isothermal amplification is a method used to synthesize and amplify DNA from RNA. Reverse transcriptase is an enzyme that reverse transcribes RNA into complementary DNA (cDNA), which is then amplified by isothermal amplification. Reverse transcriptase isothermal amplification can be used for gene expression profiling, determining gene expression, or identifying the sequence of an RNA transcription product, including transcription start and stop sites.

In some embodiments, isothermal amplification includes strand displacement amplification (SDA). SDA relies on the ability of certain restriction enzymes to nick DNA and the ability of a 5'-3' exonuclease-deficient polymerase to extend and displace the downstream strand. Exponential nucleic acid amplification can be achieved by combining sense and antisense reactions, with strand displacement from the sense reaction serving as a template for the antisense reaction. For example, a nickase enzyme can be used that does not cleave DNA but creates a nick in one of the DNA strands, such as Nt. Alwl. SDA can also include a combination of a thermostable restriction enzyme (e.g., Aval) and a thermostable polymerase (e.g., Bst polymerase). Such a combination is known to increase amplification efficiency by approximately 10-fold, thus allowing amplification of target nucleic acids present as a single copy in the test sample.

In some embodiments, the amplification reaction assay includes transcription-mediated amplification (TMA) or nucleic acid sequence-based amplification (NASBA). In TMA and NASBA, RNA polymerase is used to amplify RNA sequences. In general, the method utilizes a first and second primer and two or three enzymes (e.g., RNA polymerase, reverse transcriptase, and optionally RNase H), as well as a first primer with a promoter sequence for the RNA polymerase. In the first step of target nucleic acid amplification, the first primer hybridizes to the target RNA (e.g., ribosomal RNA) at a defined site. Reverse transcriptase creates a cDNA copy of the target rRNA by extension from the 3' end of the promoter primer. The resulting RNA:DNA duplex RNA can be degraded by the RNase activity of reverse transcriptase (if present) or additional RNase H. A second primer then binds to the cDNA copy and a new strand of DNA is synthesized from the end of this primer by reverse transcriptase, creating a double-stranded DNA molecule. RNA polymerase recognizes the promoter sequence of the DNA template and initiates transcription. Each newly synthesized RNA amplicon re-enters the process described above and serves as a template for a new round of replication.

In some embodiments, the amplification reaction comprises recombinase polymerase amplification (RPA). Thus, isothermal amplification of a target nucleic acid is achieved by binding of opposing oligonucleotide primers to a template nucleic acid and extension of the primers by a polymerase (see, e.g., Piepenburg et al., (2006) PloS Biol., 4(7); e204). In some cases, the isothermal amplification reaction comprises a recombinase (e.g., RecA from bacteria or UvsX from bacteriophage T4), one or more cofactors (e.g., ATP or UvsY), and/or one or more single-stranded binding proteins (SSB).

In some embodiments, the isothermal amplification assay comprises helicase-dependent amplification (HDA). HDA mimics an in vivo system that uses a DNA helicase enzyme to generate a single-stranded template for primer hybridization and subsequent primer extension by a DNA polymerase. In the first step of the HDA reaction, the helicase enzyme moves along the target nucleic acid, creating a single-stranded target region that allows a primer to anneal to the target region. Next, a DNA polymerase uses free deoxyribonucleoside triphosphates (dNTPs) to extend the 3' end of each primer, generating two DNA copies. The two replicated DNA strands independently enter the next cycle of HDA, resulting in exponential nucleic acid amplification of the target nucleic acid.

In some embodiments, the isothermal amplification assay comprises rolling circle amplification (RCA). Typically, in RCA, a polymerase extends a primer continuously around a circular template, generating a long amplification product that contains many repeated copies of the circular template. By the end of the reaction, the polymerase generates thousands of copies of the circular template, with the copy strands tethered to the original target nucleic acid. RCA allows for spatial resolution and rapid nucleic acid amplification of the target nucleic acid. Branched amplification is a variation of RCA that utilizes a closed circular probe (C-probe) or padlock probe and a highly processive polymerase to exponentially amplify the C-probe under isothermal conditions.

In some embodiments, the isothermal amplification assay comprises loop-mediated isothermal amplification (LAMP). LAMP provides selectivity and employs a polymerase and a set of specifically designed primers that recognize distinct sequences in the target nucleic acid (see, e.g., Nixon et al., (2014) Bimolecular Detection and Quantitation, 2:4-10; Schuler et al., (2016) Anal Methods., 8:2750-2755; and Schoepp et al., (2017) Sci. Transl. Med., 9:eaal3693). Unlike PCR, the target nucleic acid is amplified at a constant temperature (e.g., 60-65°C) using multiple inner and outer primers and a polymerase with strand displacement activity. In some cases, an inner primer pair containing a nucleic acid sequence complementary to a portion of the sense and antisense strands of the target nucleic acid initiates LAMP. Following strand displacement synthesis by the inner primer, strand displacement synthesis primed by the outer primer pair can cause the release of a single-stranded amplicon. The single-stranded amplicon can serve as a template for further synthesis primed by a second inner and second outer primer that hybridizes to the other end of the target nucleic acid and generates a stem-loop nucleic acid structure. In subsequent LAMP cycling, one inner primer hybridizes to the loop on the product and initiates displacement and target nucleic acid synthesis to generate the original stem-loop product and a new stem-loop product with a stem that is twice as long. In addition, the 3' end of the amplicon loop structure serves as the initiation site for self-templated strand synthesis, generating a hairpin-like amplicon that forms an additional loop structure to prime subsequent rounds of self-templated amplification. Amplification continues with the accumulation of many copies of the target nucleic acid. The end product of the LAMP process is a stem-loop nucleic acid with linked repeats of the target nucleic acid in a cauliflower-like structure with multiple loops formed by annealing between alternating inverted repeats of the target nucleic acid sequence in the same strand.

In some embodiments, the isothermal amplification assay includes a digital reverse transcription loop-mediated isothermal amplification (dRT-LAMP) reaction to quantify the target nucleic acid (see, e.g., Khorosheva et al., (2016) Nucleic Acid Research, 44:2 e10). Typically, the LAMP assay generates a detectable signal (such as fluorescence) during the amplification reaction. In some embodiments, the fluorescence can be detected and quantified. Any suitable method for detecting and quantifying the fluorescence can be used. In some cases, a device such as Applied Biosystem's QuantStudio can be used to detect and quantify the fluorescence from the isothermal amplification assay.

F. Detection The present invention may be practiced using any suitable method for detecting the amplification of a target nucleic acid in a test sample by quantitative real-time isothermal amplification. In some embodiments, quantitative real-time isothermal amplification of a target nucleic acid in a test sample may be determined by detecting one or more different (distinct) fluorescent labels attached to nucleotides or nucleotide analogs incorporated during the isothermal amplification of the target nucleic acid, such as 5-FAM (522 nm), ROX (608 nm), FITC (518 nm) and Nile Red (628 nm). In another embodiment, quantitative real-time isothermal amplification of a target nucleic acid in a test sample may be determined by detection of a single fluorophore species (e.g., ROX (608 nm)) attached to nucleotides or nucleotide analogs incorporated during the isothermal amplification of the target nucleic acid. In some embodiments, each fluorophore species used emits a fluorescent signal that is distinct from the other fluorophore species, such that each fluorophore can be easily detected among other fluorophore species present in the assay.

In some embodiments, a method for detecting amplification of a target nucleic acid in a test sample by quantitative real-time isothermal amplification may include using an intercalating fluorescent dye, such as a SYTO dye (SYTO9 or SYTO82).

In some embodiments, a method for detecting amplification of a target nucleic acid in a test sample by quantitative real-time isothermal amplification may include isothermally amplifying a target nucleic acid in the test sample using an unlabeled primer and detecting the isothermal amplification of the target nucleic acid in the test sample using a labeled probe (e.g., having a fluorophore).

In some embodiments, a method for detecting amplification of a target nucleic acid in a test sample by quantitative real-time isothermal amplification may include isothermally amplifying a target nucleic acid present in a test sample using unlabeled primers and detecting isothermal amplification of the target nucleic acid using a probe having a 5-FAM dye label at the 5' end and a minor groove binder (MGB) and a non-fluorescent quencher at the 3' end (e.g., TaqMan Gene Expression Assays from ThermoFisher Scientific).

In some embodiments, detection of amplification of a target nucleic acid in a test sample can be performed using a one-stage or two-stage quantitative real-time isothermal amplification assay. In a one-stage quantitative real-time isothermal amplification assay, reverse transcription is combined with quantitative isothermal amplification to form a single quantitative real-time isothermal amplification assay. A one-stage assay reduces the number of hands-on operations and the total time to process a test sample. In some cases, for example, when a portion of the cDNA from the reverse transcription reaction is required or retained for other reactions, the quantitative real-time isothermal amplification assay can include a two-stage assay. A two-stage assay includes a first stage in which reverse transcription is performed and then a second stage in which quantitative isothermal amplification is performed. It is within the skill of one in the art to determine whether to perform a one-stage or two-stage assay.

G. Generating standard curves Because isothermal amplification assays may amplify different nucleic acids with different efficiencies, it may be necessary to calibrate the time-to-threshold value measured in real-time isothermal amplification assays to the absolute copy numbers of target and reference nucleic acids.This can be achieved by establishing a standard curve of isothermal amplification of target and reference nucleic acids.The standard curve can be obtained by performing real-time isothermal amplification assays using calibrator samples quantified at multiple known input concentrations.

In some embodiments, to generate a standard curve, quantified calibrator samples are obtained by performing serial dilutions of the quantified material. As an example, a template is prepared with a concentration of approximately 1×10 ⁹ copies/μL of target nucleic acid. To obtain calibrator samples with different input concentrations, the template is diluted in buffer at 10-fold concentration intervals to obtain templates covering a concentration range of approximately 10 ⁹ copies/μL to approximately 10 ² copies/μL. For example, eight calibrator samples are obtained with concentrations of approximately 1×10 ⁹ , 1×10 ⁸ , 1×10 ⁷ , 1×10 ⁶ , 1×10 ⁵ , 1×10 ⁴ , 1×10 ³ and 1×10 ² copies/μL, respectively. The exact concentration of each calibrator sample can be determined using methods known in the art.

To obtain a standard curve, a real-time isothermal amplification assay is performed on each aliquot containing a known amount (e.g., 1 μL) of each calibrator sample containing the respective concentration of the target nucleic acid. For example, each aliquot may contain 1 μL of each calibrator sample. For example, in the above example with eight calibrator samples covering a concentration range of 10 ⁹ copies/μL to 10 ² copies/μL, the eight aliquots of the eight calibrator samples each contain the following copy numbers of the target nucleic acid: copy number ₁ = 1×10 ⁹ , copy number ₂ = 1×10 ⁸ , copy number ₃ = 1×10 ⁷ , copy number ₄ = 1×10 ⁶ , copy number ₅ = 1×10 ⁵ , copy number ₆ = 1×10 ⁴ , copy number ₇ = 1×10 ³ , copy number ₈ = 1×10 ² .

In the real-time isothermal amplification assay for each calibrator sample, the intensity of the fluorescence emitted by intercalating fluorescent dyes (such as dsDNA dyes) or fluorescent labels for the target nucleic acid is measured as a function of time. FIG. 1 shows an exemplary plot 110 of the fluorescence intensity as a function of time in a real-time quantitative isothermal amplification assay according to some embodiments. The dashed line 120 represents a predetermined threshold intensity. The time to threshold Tt is the time elapsed from the moment the isothermal amplification is started. FIG. 2 shows an exemplary plot 210 of the fluorescence intensity as a function of time in a real-time quantitative isothermal amplification assay for eight samples with different concentrations of target nucleic acid according to some embodiments. The respective time to threshold values can be determined from each respective fluorescence curve 210 as a function of time. Thus, eight time to threshold values Tt ₁ , Tt ₂ , Tt ₃ , Tt ₄ , Tt ₅ , Tt ₆ , Tt ₇ , Tt ₈ are obtained for each of the eight calibrator samples.

For exponential amplification, the time to threshold is linearly proportional to the logarithm (e.g., log base 10) of the starting copy number (also called the template abundance ratio). FIG. 3 shows a scatter plot of data points (represented by circles) obtained from the fluorescence curves shown in FIG. 2, according to some embodiments. Each data point represents a data pair [Log ₁₀ (copy number), Tt] (note that copy number refers to the starting copy number of the nucleic acid in the isothermal amplification assay). As shown, the data points lie approximately on a straight line 310. A linear regression is performed on the data points in the plot to obtain a straight line 310 that best fits the data points with the minimum total deviation. The result of the linear regression is a straight line represented by the following equation:
Tt = m × Log _{10 (copy number) +} b (1)
where m is the slope of line 310 and b is the y-intercept. The slope m represents the efficiency of isothermal amplification of the target nucleic acid, and b represents the time to a threshold when the copy number of the template approaches zero. The line represented by equation (1) is called the standard curve.

In some embodiments, replicates (e.g., triplicates) of the isothermal amplification assay can be performed for each sample to obtain a higher level of data confidence. The replicate time-to-threshold values can be averaged and the standard deviation calculated.

Once a standard curve has been established for a given isothermal amplification assay, the standard curve can be used to convert time to threshold values to starting copy numbers for future runs of isothermal amplification assays of target nucleic acids of unknown starting copy numbers using the following equation:

Dynamic Range of the Standard Curve Usually, all low or very high copy number data points may fall outside the line 310. The range of copy numbers within which the data points can be represented by the line 310 is called the dynamic range of the standard curve. The linear relationship between the time to threshold and the logarithm of the copy number represented by the standard curve is only valid within the dynamic range.

If the amplification efficiencies of the target and reference nucleic acids differ for a given isothermal amplification assay, it may be necessary to obtain separate standard curves for the target and reference nucleic acids. Thus, two sets of real-time isothermal amplification assays can be performed, one set to establish a standard curve for the target nucleic acid and another set to establish a standard curve for the reference nucleic acid. If multiple target nucleic acids are considered, a standard curve for each target nucleic acid can be obtained.

Off-board standard curve In some embodiments, the standard curve is generated before obtaining the test sample. That is, the standard curve is not generated on-board with the quantitative isothermal amplification of the test sample. Such a standard curve may be referred to as an off-board standard curve. As described below, the off-board standard curve can be used to estimate the relative abundance value.

H. Estimation of Relative Abundance Values For a test sample with an unknown input concentration of target nucleic acid A, a first real-time isothermal amplification assay is performed on a first aliquot of the test sample to obtain a first time-to-threshold value, Tt _A , for target nucleic acid A. A second real-time isothermal amplification assay is performed on a second aliquot of the test sample to obtain a second time-to-threshold value, Tt _B , for a reference nucleic acid. The first and second aliquots contain substantially equal amounts of test sample. The time-to-first-threshold value, Tt _A , can be converted to a starting copy number of target nucleic acid A using a standard curve for target nucleic acid A:
The time value to the second threshold, Tt _B , can be converted to the starting copy number of reference nucleic acid B using a standard curve of reference nucleic acid B:
Normalizing the starting copy number of target nucleic acid A to the copy number of reference nucleic acid B gives the following relative abundance ratio:

For example, if the starting copy number of target nucleic acid A is 1000 and the starting copy number of reference nucleic acid B is 10, the relative abundance ratio (A/B) can be calculated as follows:

If the first and second aliquots contain different amounts of the test sample, the relative abundance is obtained as follows:
where _M1 and _M2 are the masses (or volumes) of the first and second aliquots, respectively.

For example, when M ₁ =1 and M ₂ =2, the starting copy number _A of the target nucleic acid is 1000, and the starting copy number _B of the reference nucleic acid is 10, the relative abundance ratio (A/B) can be calculated as follows:

In some other embodiments, one isothermal amplification assay may be performed on both target nucleic acid A and reference nucleic acid B. Because the isothermal amplification assays are performed on the same aliquot of the test sample, the relative abundance ratios can be obtained using equation (5).

Relative abundance ratio without using standard curve If the amplification efficiency of target nucleic acid and reference nucleic acid is approximately the same as known value, and the expected copy number of target nucleic acid and the expected copy number of reference nucleic acid are within the dynamic range, the relative abundance ratio can be obtained directly from the time to threshold value without using standard curve.For example, assuming that the amplification efficiency of both target nucleic acid and reference nucleic acid is m, and b is the same in both assays as a result of similar efficiency, the relative abundance ratio can be calculated as follows:

As described above, when the amplification efficiencies of the target and reference nucleic acids are approximately the same as known values, determining the relative abundance of the target nucleic acid in a test sample is not based on the starting copy number of the target nucleic acid and does not require absolute quantification of the target nucleic acid in the sample.

I. Multiple target nucleic acids In some embodiments, the relative abundance value of one target nucleic acid can be used in combination with one or more additional relative abundance values of additional target nucleic acids in test sample.In some embodiments, multiple relative abundances of multiple target nucleic acids in test sample can be used as input to an algorithm that can output a single diagnostic score, including a diagnostic score that can distinguish between viral and bacterial infections in test sample and/or diagnose that test sample has bacterial and/or viral infection (see, for example, Sweeney et al., (2016), Sci.Transl.Med., 8:346ras91346ra91).

In some embodiments, the target nucleic acid selected for evaluation is selected from target nucleic acids known to be increased in host expression as a result of bacterial infection, such as CTSB, TNIP1, GPAA1, and HK3. In some embodiments, the target nucleic acid selected for evaluation is selected from target nucleic acids known to be increased in host expression as a result of viral infection, such as IFI27, JUP, and LAX1.

In some embodiments, the genes are combined using machine learning or other algorithms to generate a single diagnostic score, such as one that can distinguish between bacterial and viral infections. Such a score may be high in patients with bacterial infections and low in patients with viral infections, such that a physician seeing a patient whose diagnostic score exceeds a set threshold would treat with antibiotics. In these embodiments, the algorithmic score has greater diagnostic power (e.g., has a greater area under the receiver operating characteristic curve for distinguishing bacterial from viral infections) than any individual gene level alone.

In some embodiments, the types of algorithms for combining multiple biomarkers into a single diagnostic score include, but are not limited to, geometric mean difference, arithmetic mean difference, sum difference, simple sum, etc. In some embodiments, the diagnostic score can be evaluated based on the relative abundance values of multiple biomarkers using machine learning models such as regression models, tree-based machine learning models, support vector machine (SVM) models, artificial neural network (ANN) models, etc.

J. Method for estimating diagnostic score by performing real-time quantitative isothermal amplification using off-board standard curve Absolute quantification usually requires a standard curve to be generated in the same assay as the test sample to ensure proper calibration and sufficient accuracy. The standard curve thus obtained is called an on-board stand curve. According to some embodiments, the method for estimating diagnostic score using real-time quantitative isothermal amplification of multiple biomarkers requires only relative quantification and therefore can use off-board standard curves. This may enable a new class of ultrafast diagnostic real-time quantitative isothermal amplification assays that can reliably and economically perform accurate diagnosis.

Figure 4 shows a simplified flowchart illustrating a method for estimating a diagnostic score by performing real-time quantitative isothermal amplification on a test sample, according to some embodiments.

At 402, a first standard curve, a second standard curve, and a reference standard curve are obtained. The first standard curve includes a first function relating the starting copy number of the first target nucleic acid to the time to a threshold value. The second standard curve includes a second function relating the starting copy number of the second target nucleic acid to the time to a threshold value. The reference standard curve includes a reference function relating the starting copy number of the reference nucleic acid to the time to a threshold value. The first standard curve, the second standard curve, and the reference standard curve are generated prior to performing real-time quantitative isothermal amplification on the test sample.

At 404, a test sample is obtained from the mammalian subject. The test sample contains a first target nucleic acid, a second target nucleic acid, and a reference nucleic acid.

At 406, the test sample is added to at least one reaction vessel. The at least one reaction vessel contains a master mix for isothermal amplification of the first target nucleic acid, the second target nucleic acid, and the reference nucleic acid.

At 408, at least one reaction of isothermal amplification is initiated in at least one reaction vessel.

At 410, a time value to a first threshold for a first target nucleic acid in at least one reaction is determined.

At 412, a time value to a second threshold for a second target nucleic acid in at least one reaction is determined.

At 414, the time to a reference threshold for the reference nucleic acid in at least one reaction is determined.

At 416, the first function of the first standard curve is used to estimate a first starting copy number of the first target nucleic acid in the test sample based on the time value to the first threshold value.

At 418, a second function of the second standard curve is used to estimate a second starting copy number of the second target nucleic acid in the test sample based on the time value to the second threshold value.

At 420, a reference starting copy number of the reference nucleic acid in the test sample is estimated based on the time value to the reference threshold using a reference function provided by the reference standard curve.

At 422, a first relative abundance value of the first target nucleic acid in the test sample relative to the reference nucleic acid is estimated based on the first starting copy number of the first target nucleic acid and the reference starting copy number of the reference nucleic acid.

At 424, a second relative abundance value of the second target nucleic acid in the test sample relative to the reference nucleic acid is estimated based on the second starting copy number of the second target nucleic acid and the reference starting copy number of the reference nucleic acid.

At 426, a diagnostic score for the test sample is estimated based on the first relative abundance value of the first target nucleic acid and the second relative abundance value of the second target nucleic acid.

At 428, a clinical diagnosis of the disease state is made by comparing the diagnostic score of the test sample to a predetermined threshold diagnostic score.

In some embodiments, the isothermal amplification is loop-mediated isothermal amplification (LAMP).

In some embodiments, at least one reaction of the isothermal amplification in at least one reaction vessel is initiated using a hot start mechanism.

In some embodiments, the diagnostic score is related to the difference between a time value to a first threshold for a first target nucleic acid and a time value to a second threshold for a second target nucleic acid.

It should be appreciated that the specific steps illustrated in FIG. 4 provide a particular method of estimating a diagnostic score by performing real-time quantitative isothermal amplification according to some embodiments of the present invention. Other sequences of steps may be performed according to alternative embodiments. For example, alternative embodiments of the present invention may perform the steps outlined above in a different order. Additionally, individual steps illustrated in FIG. 4 may include multiple sub-steps that may be performed in various sequences appropriate to the individual step. Additionally, additional steps may be added or removed depending on the particular application. Those skilled in the art will recognize numerous variations, modifications, and alternatives.

K. Methods for Estimating Diagnostic Score by Performing Real-Time Quantitative Isothermal Amplification Without Using a Standard Curve According to some embodiments, the methods for estimating diagnostic score can utilize real-time quantitative isothermal amplification without using a standard curve. For example, the diagnostic score can integrate the expression levels of multiple previously identified biomarkers. If the isothermal amplification assays of the identified biomarkers are pre-validated across a target test population to be expected to perform within a linear dynamic range, the time-to-threshold values can be plugged directly into an algorithm for estimating diagnostic score without being converted to copy number using a standard curve. In some embodiments, a clinically relevant threshold diagnostic score can be established by a population study. In a population study, a real-time quantitative isothermal amplification assay is performed across a clinical cohort of patients of interest. The time-to-threshold values obtained from the real-time quantitative isothermal amplification assay are used to train a statistical model for establishing a threshold diagnostic score. Once the threshold diagnostic score is established, it can be used to diagnose patients.

Figure 5 shows a simplified flowchart illustrating a method for estimating a diagnostic score by performing real-time quantitative isothermal amplification of a test sample without the use of a standard curve, according to some other embodiments.

At 502, a test sample is obtained from a mammalian subject. The test sample contains at least one first target nucleic acid, at least one second target nucleic acid, and a reference nucleic acid. Each of the first target nucleic acid, the second target nucleic acid, and the reference nucleic acid has an expected concentration in the test sample that is within the dynamic range of the real-time quantitative isothermal amplification, as validated across a cohort population of interest.

At 504, an aliquot of the test sample is added to at least one reaction vessel containing a master mix for isothermal amplification of the first target nucleic acid, the second target nucleic acid, and the reference nucleic acid.

At 506, at least one reaction of isothermal amplification is initiated in at least one reaction vessel.

At 508, a time value to a first threshold value for the first target nucleic acid in at least one reaction is determined.

At 510, a time value to a second threshold for a second target nucleic acid in at least one reaction is determined.

At 512, a time value to a reference threshold value for the reference nucleic acid in at least one reaction is determined.

At 514, a first relative abundance value of the first target nucleic acid relative to the reference nucleic acid in the test sample is estimated based on at least the time value to the first threshold and the time value to the reference threshold.

At 516, a second relative abundance value of the second target nucleic acid relative to the reference nucleic acid in the test sample is estimated based on at least the time value to the second threshold and the time value to the reference threshold.

At 518, a diagnostic score for the test sample is estimated based on the time value to the first threshold for the first target nucleic acid and the time value to the second threshold for the second target nucleic acid.

In some embodiments, the method 500 further includes performing real-time quantitative isothermal amplification across a cohort population of interest prior to obtaining a test sample to establish a clinically relevant threshold diagnostic score, and after estimating the diagnostic score of the test sample, making a clinical diagnosis of the disease state by comparing the diagnostic score of the test sample to the threshold diagnostic score.

In some embodiments, the isothermal amplification is loop-mediated isothermal amplification (LAMP).

In some embodiments, the diagnostic score is related to the difference between a time value to a first threshold for a first target nucleic acid and a time value to a second threshold for a second target nucleic acid.

In some embodiments, the test sample contains a plurality of first target nucleic acids and a plurality of second target nucleic acids. The diagnostic score is related to the difference between a first statistical value based on the time to threshold values of the plurality of first target nucleic acids and a second statistical value based on the time to threshold values of the plurality of second target nucleic acids.

It should be appreciated that the specific steps illustrated in FIG. 5 provide a particular method for estimating a diagnostic score by performing real-time quantitative isothermal amplification according to some embodiments of the present invention. Other sequences of steps may be performed according to alternative embodiments. For example, alternative embodiments of the present invention may perform the steps outlined above in a different order. Additionally, individual steps illustrated in FIG. 5 may include multiple sub-steps that may be performed in various sequences appropriate to the individual step. Additionally, additional steps may be added or removed depending on the particular application. Those skilled in the art will recognize numerous variations, modifications, and alternatives.

L. Methods for Estimating Diagnostic Scores Using Multi-Volume Real-Time Quantitative Isothermal Amplification Assays According to some embodiments, methods for estimating diagnostic scores using multiple biomarkers can utilize multi-volume real-time quantitative isothermal amplification approaches as described above. For example, in a HostDx-Fever qLAMP assay that includes seven identified target genes IFI27, JUP, LAX1, HK3, TNIP1, GPAA1, CTSB, some of the seven target genes are expected to be more abundant than some other target genes in a test sample. It may be advantageous to perform real-time quantitative isothermal amplification assays for genes that are expected to be more abundant in a reaction vessel with a large volume and genes that are expected to be less abundant in a reaction vessel with a small volume. Thus, given a limited amount of test sample, quantitative isothermal amplification assays can be successfully performed for both genes with high expected abundance and genes with low expected abundance.

In some embodiments, the expected relative abundance of various genes can be established by population testing prior to allocation to wells. In population testing, a real-time quantitative isothermal amplification assay is performed for various genes in a clinically representative population large enough to establish statistical significance. For example, population testing can use Student's t-test, Welch's t-test, Mann-Whitney U-test, or analysis of variance (ANOVA), F-test, etc. Statistical significance can be set at, for example, p<0.05.

Figure 6 shows a simplified flowchart illustrating a method for estimating a diagnostic score by performing real-time quantitative isothermal amplification on a test sample, according to some embodiments.

At 602, a test sample is obtained. The test sample contains at least one first target nucleic acid, at least one second target nucleic acid, and a reference nucleic acid. Each of the first target nucleic acid, the second target nucleic acid, and the reference nucleic acid comprises a mammalian host nucleic acid.

At 604, a first aliquot of the test sample is added to a first reaction vessel for quantitative isothermal amplification of a first target nucleic acid, and a second aliquot of the test sample is added to a second reaction vessel for quantitative isothermal amplification of a second target nucleic acid. Each of the first reaction vessel and the second reaction vessel contains a master mix for isothermal amplification of a first target nucleic acid, a second target nucleic acid, and a reference nucleic acid. The second target nucleic acid has an expected lower abundance in the test sample than the first target nucleic acid. The first aliquot has a first volume. The second aliquot has a second volume that is greater than the first volume.

At 606, a first real-time quantitative isothermal amplification assay is performed in the first reaction vessel by initiating a first reaction in the first reaction vessel, determining a time value to a first threshold for a first target nucleic acid in the first reaction, determining a time value to a first reference threshold for a reference nucleic acid in the first reaction, and estimating a first relative abundance value of the first target nucleic acid in the test sample relative to the reference nucleic acid based on at least the time value to the first threshold and the time value to the first reference threshold.

At 608, a second real-time quantitative isothermal amplification assay is performed in the second reaction vessel by initiating a second reaction in the second reaction vessel, determining a time value to a second threshold for a second target nucleic acid in the second reaction, determining a time value to a second reference threshold for a reference nucleic acid in the second reaction, and estimating a second relative abundance value of the second target nucleic acid in the test sample relative to the reference nucleic acid based on at least the time value to the second threshold and the time value to the second reference threshold.

At 610, a diagnostic score for the test sample is estimated based on the first relative abundance ratio value of the first target nucleic acid and the second relative abundance ratio value of the second target nucleic acid.

In some embodiments, the method 600 further includes establishing that the second target nucleic acid has a lower expected abundance than the first target nucleic acid by performing a test real-time quantitative isothermal amplification assay for the first target nucleic acid and the second target nucleic acid in a clinically representative population prior to adding a first aliquot of the test sample to the first reaction vessel and adding a second aliquot of the test sample to the second reaction vessel.

In some embodiments, the diagnostic score is related to the difference between the first relative abundance value and the second relative abundance value.

In some embodiments, the test sample contains a plurality of first target nucleic acids and a plurality of second target nucleic acids, and the diagnostic score is related to the difference between a first statistical value based on the relative abundance values of the plurality of first target nucleic acids and a second statistical value based on the relative abundance values of the plurality of second target nucleic acids. In some embodiments, the first statistical value includes a geometric mean of the relative abundance values of the plurality of first target nucleic acids, and the second statistical value includes a geometric mean of the relative abundance values of the plurality of second target nucleic acids. In some embodiments, the diagnostic score is used to diagnose whether a patient has a bacterial infection or a viral infection. In some embodiments, the plurality of first target nucleic acids includes genes that are more prevalent in viral infections, and the plurality of second target nucleic acids includes genes that are more prevalent in bacterial infections. For example, the plurality of first target nucleic acids may include IFI27, JUP, and LAX1, and the plurality of second target nucleic acids may include HK3, TNIP1, GPAA1, and CTSB.

In some embodiments, the test sample contains a plurality of first target nucleic acids and a plurality of second target nucleic acids. The diagnostic score is estimated based on the relative abundance ratio values of the plurality of first target nucleic acids and the relative abundance ratio values of the plurality of second target nucleic acids using a regression model, a tree-based machine learning model, a support vector machine model, or an artificial neural network (ANN) model.

In some embodiments, each of the first real-time quantitative isothermal amplification assay and the second real-time quantitative isothermal amplification assay is a real-time quantitative loop-mediated isothermal amplification (LAMP) assay.

In some embodiments, each of the first reaction in the first reaction vessel and the second reaction in the second reaction vessel is initiated using a hot start mechanism.

It should be appreciated that the specific steps illustrated in FIG. 6 provide a particular method for estimating a diagnostic score by performing real-time quantitative isothermal amplification according to some embodiments of the present invention. Other sequences of steps may be performed according to alternative embodiments. For example, alternative embodiments of the present invention may perform the steps outlined above in a different order. Additionally, individual steps illustrated in FIG. 6 may include multiple sub-steps that may be performed in various sequences appropriate to the individual step. Additionally, additional steps may be added or removed depending on the particular application. Those skilled in the art will recognize numerous variations, modifications, and alternatives.

M. Platform/Device In some aspects, the present disclosure provides a platform or device for obtaining the relative abundance ratio of target nucleic acid in a test sample. Any suitable device or platform for carrying out the method can be used. In some embodiments, the device is useful for comparatively quantifying host mammalian nucleic acid in a test sample, where the host mammalian nucleic acid reflects a host having an acute infection, such as a bacterial or viral infection. In some embodiments, the acute infection induces sepsis.

In some embodiments, the device is a microfluidic device. In some embodiments, the microfluidic device allows for amplification of the target nucleic acid and the reference nucleic acid in the same reaction vessel. In other embodiments, the microfluidic device allows for amplification of the target nucleic acid and the reference nucleic acid in separate reaction vessels (such as separate wells).

Any device that can heat a sample to a certain temperature and monitor fluorescence, turbidity, luminescence, absorbance (color) or magnetic/electromagnetic current can be used for isothermal amplification. In some embodiments, the device is ThermoFisher Scientific's TaqMan Gene Expression Assay.

In some embodiments, devices for carrying out the method include fluorescent label detection systems, such as, but not limited to, Applied Biosystem's QuantStudio real-time PCR system (under isothermal conditions).

As will be apparent to one of skill in the art, the platform or device used to practice the present invention can include any useful dimensions (such as length, width, and depth). In some embodiments, the device is a bench-top sized device that utilizes one or more reaction vessels containing the target and reference nucleic acids. In some embodiments, the reaction vessels are housed in a single unit, such as a 96-well plate. In some embodiments, the reaction vessels (or multiple housing units) can be stored, tested, and/or analyzed sequentially or simultaneously within the device.

Figure 7 shows a schematic block diagram of an apparatus 700 for estimating relative abundance values using a real-time quantitative isothermal amplification assay, according to some embodiments.

The apparatus 700 includes one or more reaction vessels 710. Each reaction vessel 710 is configured to hold an aliquot of a test sample containing at least one target nucleic acid and a reference nucleic acid. Each reaction vessel 710 is further configured to hold a master mix for isothermal amplification of the at least one target nucleic acid and the reference nucleic acid. The master mix also includes a fluorescent label for detecting the at least one target nucleic acid and the reference nucleic acid.

In some embodiments, the one or more reaction vessels 710 include at least a first reaction vessel and a second reaction vessel. The first reaction vessel may be configured to hold a first aliquot of the test sample and a first portion of a master mix for isothermal amplification of a first target nucleic acid. The second reaction vessel may be configured to hold a second aliquot of the test sample and a second portion of a master mix for isothermal amplification of a second target nucleic acid. In some embodiments, the one or more reaction vessels 710 include a third reaction vessel configured to hold a third aliquot of the test sample and a third portion of a master mix for isothermal amplification of a reference nucleic acid.

The apparatus 700 further includes an isothermal amplification means 720 for initiating an isothermal amplification reaction in one or more reaction vessels 710. For example, the isothermal amplification means 720 may include a heating element and temperature control for heating the reaction vessel 710 and its contents to a temperature necessary to initiate isothermal amplification of at least one target nucleic acid and a reference nucleic acid. The isothermal amplification reaction may generate fluorescence associated with at least one target nucleic acid and fluorescence associated with the reference nucleic acid.

The apparatus 700 further includes one or more fluorescence detectors 730 optically coupled to the one or more reaction vessels 710. Each fluorescence detector 730 is configured to detect the fluorescence associated with the respective target nucleic acid or the fluorescence associated with the reference nucleic acid in real time during the isothermal amplification reaction. Thus, the intensity of the fluorescence associated with the respective target nucleic acid can be measured as a function of time, and the intensity of the fluorescence associated with the reference nucleic acid can be measured as a function of time. In some embodiments, each fluorescence detector is configured to detect the fluorescence associated with the respective target nucleic acid at regular intervals during the isothermal amplification reaction. For example, the regular intervals of fluorescence detection can be performed once every minute, once every 30 seconds, once every 20 seconds, once every 10 seconds, once every 5 seconds, or once every second during the isothermal amplification reaction.

The device 700 further includes a computer memory 740. In some embodiments, the computer memory is configured to store one or more standard curves. For example, the first standard curve may provide a first function relating the starting copy number of the first target nucleic acid to the time to a threshold value. The second standard curve may provide a second function relating the starting copy number of the second target nucleic acid to the time to a threshold value. The third standard curve may provide a third function relating the starting copy number of the reference nucleic acid to the time to a threshold value. The first standard curve, the second standard curve, and the third standard curve may be obtained from a previous isothermal amplification reaction of the calibrator sample and stored in the memory 740 for use in a subsequent isothermal amplification reaction of the test sample. In some embodiments, the computer memory 740 is configured to store one or more threshold fluorescence intensity values.

The apparatus 700 further includes a computer processor 750 coupled to the fluorescence detector 730 and the memory 740. The memory 740 can further store instructions executed by the computer processor 750.

In some embodiments, the processor 750 is configured to determine a first threshold time value for the first target nucleic acid based on the intensity of the fluorescence associated with the first target nucleic acid as a function of time and the stored first threshold fluorescence intensity value, and estimate the starting copy number of the first target nucleic acid in the test sample based on the time value to the first threshold using a first function provided by the first standard curve. The processor 750 is further configured to determine a second threshold time value for the second target nucleic acid based on the intensity of the fluorescence associated with the second target nucleic acid as a function of time and the stored second threshold fluorescence intensity value, and estimate the starting copy number of the second target nucleic acid in the test sample based on the time value to the second threshold using the first function provided by the second standard curve. The processor 750 is further configured to determine a third threshold time value for the reference nucleic acid based on the intensity of the fluorescence associated with the reference nucleic acid as a function of time and the stored third threshold fluorescence intensity value, and estimate the starting copy number of the reference nucleic acid in the test sample based on the time value to the third threshold using a third function provided by the third standard curve. The processor 750 is further configured to estimate a relative abundance value of the first target nucleic acid in the test sample relative to the reference nucleic acid based on the starting copy number of the first target nucleic acid and the starting copy number of the reference nucleic acid, and to estimate a relative abundance value of the second target nucleic acid in the test sample relative to the reference nucleic acid based on the starting copy number of the second target nucleic acid and the starting copy number of the reference nucleic acid. The processor 750 may be configured to estimate a diagnostic score for the test sample based on the first relative abundance value of the first target nucleic acid and the second relative abundance value of the second target nucleic acid.

N. Kits In some aspects, the disclosure provides kits for calculating relative abundance values of target nucleic acids in test samples. In some embodiments, the kits include at least one reaction vessel and one or more components for performing real-time quantitative isothermal amplification (e.g., RNA polymerase, reverse transcriptase, and/or DNA polymerase). In some embodiments, the kits can include two or more reaction vessels (e.g., a first reaction vessel and a second reaction vessel). In some embodiments, the kits include a master mix for isothermal amplification. In some embodiments, the kits include a master mix for real-time quantitative isothermal amplification. The kits may come with instructions for interpreting results from a real-time quantitative isothermal amplification assay. Instructions (written, CD-ROM, etc.) for performing a real-time quantitative isothermal amplification assay may also be included in the kit.

The following examples are provided to illustrate, but not to limit, the claimed invention.

Example 1: Isothermal Amplification Assay Configuration The isothermal assay primers include a forward inner primer (FIP) and a forward outer primer (F3) and the corresponding backward inner primer (BIP) and backward outer primer (B3), along with forward and backward rate enhancing primers (FR, BR). The assay is performed using WarmStart (also called hot start) LAMP 2X Master Mix (NEB, CAT#E1700S) following the manufacturer's protocol adjusted to a total reaction volume of 20 μL and adding any fluorescent dye to a final concentration of 1×. Primers are added at final concentrations of 1.6 μM FIP/BIP, 0.2 μM F3/B3, and 0.4 μM FR/BR. To improve assay fidelity, the assay is supplemented with 1 mM dUTP (ThermoFisher CAT#R0133). Template is added to a standard 1 μL volume containing an empirically optimized mass of sample material. Finally, water is added to bring the final volume per reaction to 20 μL. The assay is dispensed into 96-well plates (ThermoFisher CAT# 4346906) for quantitative amplification using a real-time PCR instrument.

Quantitative isothermal amplification was performed on a QuantStudio 6 Flex real-time PCR system (ThermoFisher) using the FAM/SYBR Green channel to monitor dye fluorescence. Cycling conditions included a 5 min hold step at 25°C followed by a 60 min hold step at 65°C during which fluorescence was monitored at 20 s intervals. The use of the NEB kit containing the warm start polymerase ensures that the isothermal reaction begins only when the solution temperature reaches 45°C. This allows for comparison of assays across plates/experiments by ensuring that equivalent time to threshold (Tt) values determined in different runs represent equal time lapses from reaction initiation across those runs. This also allows the assay to be calibrated to a previously determined standard curve so that assays of varying efficiency can be compared for relative target quantification.

Example 2: Obtaining a standard curve and calculating the relative abundance ratio To calibrate the isothermal amplification assay, a double-stranded DNA template corresponding to the mRNA sequence of interest is synthesized by a commercial vendor (such as Integrated DNA Technologies) to be used as a quantified control sample. The lyophilized synthetic template is resuspended in TE buffer {20 mM Tris (pH 8.0), 0.5 mM EDTA} to a final concentration of approximately 1 x ¹⁰⁹ copies/μL. The exact concentration of each template is determined using a Qubit fluorometer and the associated Qubit dsDNA HS assay kit (such as ThermoFisher Scientific, CAT# Q32851) according to the manufacturer's protocol. This value is used in the subsequent regression analysis. To generate samples for standard curve analysis, each template was diluted in TE buffer at 10-fold concentration intervals to obtain samples covering the range of approximately ¹⁰ copies/μL to approximately ¹⁰ copies/μL. The time to threshold (Tt) for each sample was determined in triplicate using 1 μL of input as template for the corresponding isothermal amplification assay.

Example 3: Selection of threshold intensity The threshold is a function of the fluorescent dye or probe used and the instrument that detects fluorescence. As a general rule, the threshold is determined by identifying the point in the linear phase of amplification that is significantly above background signal and maintains the minimum standard deviation of the resulting time to threshold (Tt) across multiple experimental repetitions. The same threshold should be used for a given target both when determining standard curves and when measuring target abundance in samples of interest. When determining a unique standard curve for each target, it is not necessary to use the same threshold for different targets, but for relative comparisons without standard curve calibration, it is important to maintain the same threshold for all targets.

Example 4: Relative quantification of target nucleic acids by isothermal amplification and comparison with gold standard mRNA quantification assays To evaluate the performance of relative mRNA quantification from patient blood samples using the isothermal amplification assays described herein, the relative abundance ratio values of the target nucleic acid (herein referred to as "biomarker IFI27") relative to a reference nucleic acid (herein referred to as "housekeeping gene YWHAB") were determined and compared to measurements obtained using a gold standard mRNA quantification assay performed on a NanoString nCounter (NanoString Technologies Inc., Seattle). NanoString is highly accurate and is a useful tool for measuring the expression levels of multiple genes at once. However, it may be too slow for clinical applications (4-6 hours per assay).

Sample Selection and RNA Extraction To approximate the range of target nucleic acid abundances expected in a clinical setting, samples were selected from patients presenting with viral infections (6) or bacterial sepsis (8) as well as a set of healthy controls (6) (see Table 1). Samples were collected into PAXgene Blood RNA Tubes (PreAnalytiX, GmbH), processed according to the manufacturer's protocol, and then stored at -80°C.

At the time of analysis, samples were thawed at room temperature for 2 h and then inverted >10 times to homogenize the contents of the vacutainers. Final RNA purification was performed on an aliquot of each sample using a Qiacube (Qiagen, MD), and purified total RNA was quantified using a Qubit fluorometer (ThermoFisher Scientific, Waltham) (see Table 1).

Results - Assessment of Relative Abundance To assess the relative abundance of target nucleic acids relative to reference nucleic acids, each target or reference nucleic acid was singleplex amplified from 50 ng of total RNA using the one-step reverse transcription-isothermal amplification assay described above.

Time to threshold (Tt) values were determined for each target and reference nucleic acid in each sample and then converted to _Log2 (template copy number) using the previously determined standard curve. The relative abundance of IFI27 to YWHAB was then calculated by taking the ratio of the two measurements (see Table 1).

The same RNA samples were also analyzed in a NanoString nCounter, which allows direct quantification of the host mRNA transcripts present in the sample without amplification. The abundance ratio values of IFI27 and YWHAB transcripts obtained using the NanoString nCounter were log transformed and the ratio of these values was determined and compared to measurements obtained using the isothermal amplification assay described above.

To quantitate the performance of the isothermal amplification assay against the gold standard, the relative abundance values determined using the isothermal amplification assay were plotted as a function of the values determined using the NanoString nCounter, and the Pearson correlation coefficient between these measurements across all samples was calculated (see FIG. 7).

Figure 8 shows a correlation plot with the Pearson coefficient relating the abundance of the target nucleic acid (IFI27) to the abundance of the reference nucleic acid (YWHAB) as determined by the isothermal amplification assay and the gold standard NanoString nCounter. Values are plotted as _Log2 (fold abundance) for both assays and are coded according to diagnosis: circles represent viral infections and squares represent bacterial sepsis. Triangles represent healthy controls. Lines represent curve fits by standard linear regression analysis for ease of interpretation.

The isothermal amplification assay demonstrated exceptional correlation with the gold standard assay (r=0.97) with excellent agreement in relative abundance measurements. Importantly, groups representing different diagnoses (e.g., bacterial sepsis, viral infections, or healthy samples) clustered together in both assays, indicating that clinical utility is maintained using the isothermal amplification assay.

Example 5: HostDx-Fever qLAMP Assay and Comparison with Gold Standard mRNA Quantitation Assay A set of seven genes was previously identified as useful biomarkers for classifying viral or bacterial infections. The seven genes include IFI27, JUP, LAX1, which are more prevalent in viral infections, and HK3, TNIP1, GPAA1, CTSB, which are more prevalent in bacterial infections. (See US Patent Application Publication No. 2019/0144943.) A combined diagnostic score based on the levels of the seven biomarkers (called the Fever Score or Host-Dx-Fever Score) can be estimated using the difference in geometric means (DGM), as expressed according to the following formula:

In the experiment, the HostDx-Fever qLAMP assay was validated for each of the seven target genes across the assay validation cohort. Total RNA was extracted and LAMP assays were run in triplicate on a QuantStudio6 qPCR machine. The analytical gold standard was NanoString nCounter absolute mRNA counts. For both techniques, the HostDx-Fever score was calculated as the DGM of the mRNA assay, as expressed in equation (8):

Relative quantification using isothermal amplification without a standard curve In the experiment, the same bacterial and viral clinical samples were tested with two sets of assays. Each set of assays targeted seven previously identified biomarkers that, when combined using the DGM approach, create a single diagnostic score that can separate bacterial and viral infections. In the first set of assays, the NanoString nCounter digital mRNA platform was used, where housekeeper-normalized numbers are input into the DGM equation (e.g., equation (8)).

The second set of assays was a set of isothermal qLAMP assays targeting the same mRNAs. The time to threshold values, Tt, for each assay were entered into the DGM equation (e.g., equation (8)). Note that the time to threshold values were entered directly into the DGM equation without being converted to absolute copy numbers using a standard curve.

Figure 9A shows a correlation plot with the Pearson coefficient associated with the diagnostic score determined by the isothermal amplification assay and the gold standard NanoString nCounter. Black circles represent viral infections and grey circles represent bacterial infections. The lines represent standard linear regression curve fits for ease of interpretation. The isothermal amplification assay demonstrated exceptional correlation with the gold standard assay (r=0.944). This indicates that even without the use of a standard curve, the qLAMP technology can provide highly accurate diagnostic results in relevant clinical samples against the analytical gold standard.

Figure 9B shows the HostDx-Fever score distribution based on the qLAMP assay. Figure 9C shows the HostDx-Fever score distribution based on the NanoStringCounter. Note that groups representing different diagnoses (e.g., bacterial or viral infections) cluster together in either type of assay. This indicates that HostDx-Fever performed with either technique can accurately distinguish between infection classes and that the HostDx-Fever biomarker is relatively insensitive to the underlying quantitative technique. However, optimal clinical cutoffs may differ for the two techniques, as the technical platforms may yield different absolute numbers while maintaining similar relative quantification.

Example 6: Multi-Volume Isothermal qLAMP Assay Experiments were performed to test whether probing larger volumes at a given template concentration could improve the success rate of qLAMP assays at the lower end of the linear dynamic range. The hypothesis is: (i) qLAMP, like qPCR, measures the time required to accumulate an arbitrary but fixed threshold number of copies of a target gene in a reaction vessel. (ii) Under saturating binding conditions, the time required to reach the threshold number depends on (a) the number of copies present when the reaction is initiated, and (b) the rate of increase per unit time (called the reaction efficiency or amplification efficiency).

In the experiment, the same concentration of template is maintained across two different volumes (10 μL and 50 μL). The isothermal amplification is considered successful if the measured time to threshold value Tt is below a predefined cutoff value. The cutoff value can be defined as the y-intercept of a linear regression fit to the standard curve titration, as exemplified below, or by other means.

Figure 10 shows an example where the cutoff value is defined as the y-intercept of a linear regression fit to the standard curve titration. The circles 1010 are the measured time to threshold values for various input concentrations. The straight line 1020 represents the standard curve obtained by the linear regression fit. The horizontal dashed line 1030 indicates the y-intercept (e.g., the "b" value of the standard curve represented in Equation (1) or Equation (2)). Thus, in some embodiments, if the measured time to threshold value Tt is below the horizontal dashed line 930, the isothermal amplification can be considered successful.

In an experiment, on a 96-well plate, 48 wells are filled with a first aliquot of 10 μL of the test sample, and the other 48 wells are filled with a second aliquot of 50 μL of the test sample. The template concentrations of the first and second aliquots are the same.

Figure 11 shows experimental results of the qLAMP assay. The solid circles represent the time to threshold Tt measured in a 20 μL reaction volume, and the grey circles represent the time to threshold Tt values measured in a 50 μL reaction volume. The inset shows an expanded version of the time to threshold range from 20 to 40 time cycles (20 seconds each). The horizontal dashed line indicates the predefined cutoff Tt value, as previously described. As shown, the larger the reaction volume, the faster the time to threshold value and the higher the success rate.

This experiment demonstrates that for a fixed number of template molecules distributed in a given volume, when the fixed number is near the lower limit of the linear dynamic range, probing a larger portion of the volume is more likely to produce an accurate (linear) measurement. Thus, using a larger well volume to assay low-abundance biomarkers may improve the linear dynamic range and increase the number of samples that can be successfully run in the assay.

It is understood that the examples and embodiments described herein are for illustrative purposes only, and that various modifications or changes will be suggested to those skilled in the art in light thereof, and are within the spirit and scope of this application and the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.

Claims (17)

1. A method for estimating a diagnostic score of a test sample using real-time quantitative isothermal amplification, the test sample containing at least one first target nucleic acid, at least one second target nucleic acid, and a reference nucleic acid, each of the first target nucleic acid, the second target nucleic acid, and the reference nucleic acid comprising a mammalian host nucleic acid, the method comprising:
adding a first aliquot of the test sample to a first reaction vessel for quantitative isothermal amplification of the first target nucleic acid and a second aliquot of the test sample to a second reaction vessel for quantitative isothermal amplification of the second target nucleic acid, wherein the first reaction vessel and the second reaction vessel each contain a master mix for isothermal amplification of the first target nucleic acid, the second target nucleic acid and the reference nucleic acid, the second target nucleic acid having a lower expected abundance in the test sample than the first target nucleic acid, the first aliquot having a first volume and the second aliquot having a second volume greater than the first volume;
conducting a first real-time quantitative isothermal amplification assay in said first reaction vessel;
initiating a first reaction in the first reaction vessel;
determining a time value to a first threshold for the first target nucleic acid in the first reaction;
determining a time value to a first reference threshold value for the reference nucleic acid in the first reaction; and
by estimating a first relative abundance value of the first target nucleic acid in the test sample relative to the reference nucleic acid based on at least the first threshold value and the first reference threshold value;
conducting a second real-time quantitative isothermal amplification assay in said second reaction vessel;
initiating a second reaction in the second reaction vessel;
determining a time value to a second threshold for the second target nucleic acid in the second reaction;
determining a time value to a second reference threshold value for the reference nucleic acid in the second reaction; and
by estimating a second relative abundance value of the second target nucleic acid in the test sample relative to the reference nucleic acid based on at least the time to second threshold value and the time to second reference threshold value;
and estimating the diagnostic score for the test sample based on the first relative abundance value of the first target nucleic acid and the second relative abundance value of the second target nucleic acid. The method of claim 1, further comprising establishing that the second target nucleic acid has a lower expected abundance than the first target nucleic acid by performing a test real-time quantitative isothermal amplification assay for the first target nucleic acid and the second target nucleic acid in a clinically representative population prior to adding the first aliquot of the test sample to the first reaction vessel and adding the second aliquot of the test sample to the second reaction vessel. The method of claim 1, wherein the diagnostic score is related to the difference between the first relative abundance value and the second relative abundance value. The method of claim 1, wherein the test sample contains a plurality of first target nucleic acids and a plurality of second target nucleic acids, and the diagnostic score is related to the difference between a first statistical value based on the relative abundance ratio values of the plurality of first target nucleic acids and a second statistical value based on the relative abundance ratio values of the plurality of second target nucleic acids. 5. The method of claim 4, wherein the first statistical value comprises a geometric mean of the relative abundance ratio values of the plurality of first target nucleic acids, and the second statistical value comprises a geometric mean of the relative abundance ratio values of the plurality of second target nucleic acids. 5. The method of claim 4, wherein the first statistical value comprises an arithmetic mean or sum of the relative abundance ratio values of the plurality of first target nucleic acids, and the second statistical value comprises an arithmetic mean or sum of the relative abundance ratio values of the plurality of second target nucleic acids. The method of claim 4, wherein the diagnostic score is used to diagnose whether a patient has a bacterial or viral infection. The method of claim 7, wherein the plurality of first target nucleic acids includes genes that are more prevalent in viral infections and the plurality of second target nucleic acids includes genes that are more prevalent in bacterial infections. The method of claim 8, wherein the plurality of first target nucleic acids includes IFI27, JUP and LAX1, and the plurality of second target nucleic acids includes HK3, TNIP1, GPAA1 and CTSB. The method of claim 1, wherein the test sample contains a plurality of first target nucleic acids and a plurality of second target nucleic acids, and the diagnostic score is estimated based on the relative abundance ratio values of the plurality of first target nucleic acids and the relative abundance ratio values of the plurality of second target nucleic acids using a regression model, a tree-based machine learning model, a support vector machine model, or an artificial neural network (ANN) model. The method of claim 1, wherein each of the first real-time quantitative isothermal amplification assay and the second real-time quantitative isothermal amplification assay is a real-time quantitative loop-mediated isothermal amplification (LAMP) assay. The method of claim 1, wherein each of the first reaction in the first reaction vessel and the second reaction in the second reaction vessel is initiated using a hot start mechanism. 1. An apparatus for estimating a diagnostic score of a test sample using real-time quantitative isothermal amplification, comprising:
a first reaction vessel configured to hold a first aliquot of the test sample, and a second reaction vessel configured to hold a second aliquot of the test sample,
the test sample contains a first target nucleic acid, a second target nucleic acid, and a reference nucleic acid, each of the first target nucleic acid, the second target nucleic acid, and the reference nucleic acid comprising a mammalian host nucleic acid;
each of the first reaction vessel and the second reaction vessel contains a master mix for isothermal amplification of the first target nucleic acid, the second target nucleic acid, and the reference nucleic acid;
a first reaction vessel and a second reaction vessel, the first aliquot having a first volume, the second aliquot having a second volume greater than the first volume, and the second target nucleic acid having a lower expected abundance in the test sample than the first target nucleic acid;
a computer memory for storing a first threshold fluorescence intensity value and a second threshold fluorescence intensity value;
means for initiating a first isothermal amplification reaction in the first reaction vessel that amplifies the first target nucleic acid and the reference nucleic acid, the first isothermal amplification reaction generating a first fluorescence associated with the first target nucleic acid and a first reference fluorescence associated with the reference nucleic acid;
a first fluorescence detector and a first reference fluorescence detector optically coupled to the first reaction vessel, the first fluorescence detector configured to measure an intensity of the first fluorescence as a function of time and to measure a first reference intensity of the first reference fluorescence as a function of time;
means for initiating a second isothermal amplification reaction in the second reaction vessel that amplifies the second target nucleic acid and the reference nucleic acid, the second isothermal amplification reaction generating a second fluorescence associated with the second target nucleic acid and a second reference fluorescence associated with the reference nucleic acid;
a second fluorescence detector and a second reference fluorescence detector optically coupled to the second reaction vessel, the second fluorescence detector configured to measure an intensity of the second fluorescence as a function of time and to measure a second reference intensity of the second reference fluorescence as a function of time;
a computer processor coupled to the first fluorescence detector, the second fluorescence detector, the first reference fluorescence detector, the second reference fluorescence detector, and the computer memory,
determining a first threshold time value for the first target nucleic acid based on the intensity of the first fluorescence as a function of time and the first threshold fluorescence intensity value;
determining a time to a first reference threshold value for the reference nucleic acid based on the intensity of the first reference fluorescence as a function of time and the first threshold fluorescence intensity value;
estimating a first relative abundance value of the first target nucleic acid in the test sample relative to the reference nucleic acid based on at least the time to the first threshold value and the time to the first reference threshold value;
determining a second threshold time value for the second target nucleic acid based on the intensity of the second fluorescence as a function of time and the second threshold fluorescence intensity value;
determining a time to a second reference threshold value for the reference nucleic acid based on the intensity of the second reference fluorescence as a function of time and the second threshold fluorescence intensity value;
estimating a second relative abundance value of the second target nucleic acid in the test sample relative to the reference nucleic acid based on at least the time to the second threshold value and the time to the second reference threshold value;
and a computer processor configured to estimate the diagnostic score for the test sample based on the first relative abundance value of the first target nucleic acid and the second relative abundance value of the second target nucleic acid. 14. The apparatus of claim 13, wherein the master mix comprises a first fluorescent label for detecting the first target nucleic acid, a second fluorescent label for detecting the second target nucleic acid, and a third fluorescent label for detecting the reference nucleic acid. 15. The apparatus of claim 14 , wherein each of the first fluorescent label, the second fluorescent label, and the third fluorescent label comprises a SYTO dye. The apparatus of claim 13 , wherein the first threshold fluorescence intensity value and the second threshold fluorescence intensity value are the same. The apparatus of claim 13 , wherein the first threshold fluorescence intensity value and the second threshold fluorescence intensity value are different from each other.